Revisiting the window of opportunity for co-transcriptional splicing efficiency and fidelity

RNA 2020 26:1081-1085. PMC7430680.  doi: 10.1261/rna.075895.120. 

Vahid Aslanzadeh and Jean D Beggs

We reported previously that, in budding yeast, transcription rate affects both the efficiency and fidelity of pre-mRNA splicing, especially of ribosomal protein transcripts. Here, we report that the majority of ribosomal protein transcripts with non-consensus 5' splice sites are spliced less efficiently when transcription is faster, and more efficiently with slower transcription. These results support the "window of opportunity" model, and we suggest a possible mechanism to explain these findings.


Spt5 modulates co-transcriptional spliceosome assembly in Saccharomyces cerevisiae

RNA 2019, 25:1298-1310. PMC6800482 doi: 10.1261/RNA.070425.119 

Isabella E Maudlin and Jean D  Beggs

There is increasing evidence from yeast to humans that pre-mRNA splicing occurs mainly co-transcriptionally, such that splicing and transcription are functionally coupled. Currently, there is little insight into the contribution of the core transcription elongation machinery to co-transcriptional spliceosome assembly and pre-mRNA splicing. Spt5 is a member of the core transcription elongation machinery and an essential protein, whose absence in budding yeast causes defects in pre-mRNA splicing. To determine how Spt5 affects pre-mRNA splicing, we used the auxin-inducible degron system to conditionally deplete Spt5 in Saccharomyces cerevisiae and assayed effects on co-transcriptional spliceosome assembly and splicing. We show that Spt5 is needed for efficient splicing and for the accumulation of U5 snRNPs at intron-containing genes, and therefore for stable co-transcriptional assembly of spliceosomes. The defect in co-transcriptional spliceosome assembly can explain the relatively mild splicing defect as being a consequence of the failure of co-transcriptional splicing. Co-immunoprecipitation of Spt5 with core spliceosomal proteins and all spliceosomal snRNAs suggests a model whereby Spt5 promotes co-transcriptional pre-mRNA splicing by stabilising the association of U5 snRNP with spliceosome complexes as they assemble on the nascent transcript. If this phenomenon is conserved in higher eukaryotes, it has potential to be important for co-transcriptional regulation of alternative splicing.


Extremely Rapid and Specific Metabolic Labelling of RNA In Vivo with 4-Thiouracil (Ers4tU).

J. Vis. Exp., 2019 (150). doi: 10.3791/59952

Barrass, J. D. and Beggs, J. D.

The nucleotide analogue, 4-thiouracil (4tU), is readily taken up by cells and incorporated into RNA as it is transcribed in vivo, allowing isolation of the RNA produced during a brief period of labelling. This is done by attaching a biotin moiety to the incorporated thio group and affinity purifying, using streptavidin coated beads. Achieving a good yield of pure, newly synthesized RNA that is free of pre-existing RNA makes shorter labelling times possible and permits increased temporal resolution in kinetic studies. This is a protocol for very specific, high yield purification of newly synthesized RNA. The protocol presented here describes how RNA is extracted from the yeast Saccharomyces cerevisiae. However, the protocol for purification of thiolated RNA from total RNA should be effective using RNA from any organism once it has been extracted from the cells. The purified RNA is suitable for analysis by many widely used techniques, such as reverse transcriptase-qPCR, RNA-seq and SLAM-seq. The specificity, sensitivity and flexibility of this technique allow unparalleled insights into RNA metabolism.


Tuning Degradation to Achieve Specific and Efficient Protein Depletion. 

J. Vis. Exp., 2019 (149), e59874, doi:10.3791/59874

Barrass, J. D., Mendoza-Ochoa, G. I., Maudlin, I. E., Sani, E., Beggs, J. D.

The plant auxin binding receptor, TIR1, recognizes proteins containing a specific auxin-inducible degron (AID) motif in the presence of auxin, targeting them for degradation. This system is exploited in many non-plant eukaryotes, such that a target protein, tagged with the AID motif, is degraded upon auxin addition. The level of TIR1 expression is critical; excessive expression leads to degradation of the AID-tagged protein even in the absence of auxin, whereas low expression leads to slow depletion. A β-estradiol-inducible AID system was created, with expression of TIR1 under the control of a β-estradiol inducible promoter. The level of TIR1 is tunable by changing the time of incubation with β-estradiol before auxin addition. This protocol describes how to rapidly deplete a target protein using the AID system. The appropriate β-estradiol incubation time depends on the abundance of the target protein. Therefore, efficient depletion depends on optimal timing that also minimizes auxin-independent depletion.


Mutagenesis of Snu114 domain IV identifies a developmental role in meiotic splicing.

RNA Biology 2019, online:

Amit Gautam and Jean D Beggs

Snu114, a component of the U5 snRNP, plays a key role in activation of the spliceosome. It controls the action of Brr2, an RNA-stimulated ATPase/RNA helicase that disrupts U4/U6 snRNA base-pairing prior to formation of the spliceosome’s catalytic centre. Snu114 has a highly conserved domain structure that resembles that of the GTPase EF-2/EF-G in the ribosome. It has been suggested that the regulatory function of Snu114 in activation of the spliceosome is mediated by its C-terminal region, however, there has been only limited characterisation of the interactions of the C-terminal domains. We show a direct interaction between protein phosphatase PP1 and Snu114 domain ‘IVa’ and identify sequence ‘YGVQYK’ as a PP1 binding motif. Interestingly, this motif is also required for Cwc21 binding. We provide evidence for mutually exclusive interaction of Cwc21 and PP1 with Snu114 and show that the affinity of Cwc21 and PP1 for Snu114 is influenced by the different nucleotide-bound states of Snu114. Moreover, we identify a novel mutation in domain IVa that, while not affecting vegetative growth of yeast cells, causes a defect in splicing transcripts of the meiotic genes, SPO22, AMA1 and MER2, thereby inhibiting an early stage of meiosis.


A fast and tuneable auxin-inducible degron for depletion of target proteins in budding yeast.

Yeast 2019 Jan;36(1):75-81.  doi: 10.1002/yea.3362.

Gonzalo I. Mendoza-Ochoa, J. David Barrass, Barbara R. Terlouw, Isabella E. Maudlin, Susana de Lucas, Emanuela Sani, Vahid Aslanzadeh, Jane A. E. Reid and Jean D. Beggs

The auxin-inducible degron (AID) is a useful technique to rapidly deplete proteins of interest in non-plant eukaryotes. Depletion is achieved by addition of the plant hormone auxin to the cell culture, which allows the auxin-binding receptor, TIR1, to target the AID-tagged protein for degradation by the proteasome. Fast depletion of the target protein requires good expression of TIR1 protein but, as we show here, high levels of TIR1 may cause uncontrolled depletion of the target protein in the absence of auxin. To enable conditional expression of TIR1 to a high level when required we regulated the expression of TIR1 using the ß-estradiol expression system. This is a fast-acting gene induction system that does not cause secondary effects on yeast cell metabolism. We demonstrate that combining the AID and ß-estradiol systems results in a tightly-controlled and fast auxin-induced depletion of nuclear target proteins. Moreover, we show that depletion rate can be tuned by modulating the duration of ß-estradiol pre-incubation. We conclude that TIR1 protein is a rate-limiting factor for target protein depletion in yeast and we provide new tools that allow tightly controlled, tuneable and efficient depletion of essential proteins while minimising secondary effects.



Transcription Rate Strongly Affects Splicing Fidelity and Co-transcriptionality in Budding Yeast

Genome Research 2017 published online December 18 2017 doi:10.1101/gr.225615.117

Vahid Aslanzadeh, Yuanhua Huang, Guido Sanguinetti and Jean D. Beggs


The functional consequences for alternative splicing of altering the transcription rate have been the subject of intensive study in mammalian cells but less is known about effects on splicing of changing the transcription rate in yeast. We present several lines of evidence showing that slow RNA polymerase II elongation increases both co-transcriptional splicing and splicing efficiency and faster elongation reduces co-transcriptional splicing and splicing efficiency in budding yeast, suggesting that splicing is more efficient when co-transcriptional. Moreover, we demonstrate that altering RNA polymerase II elongation rate in either direction compromises splicing fidelity, and we reveal that splicing fidelity depends largely on intron length together with secondary structure and splice site score. These effects are notably stronger for the highly expressed ribosomal protein coding transcripts. We propose that transcription by RNA polymerase II is tuned to optimise the efficiency and accuracy of ribosomal protein gene expression, while allowing flexibility in splice site choice with the non-ribosomal protein transcripts.


Extremely fast and incredibly close: co-transcriptional splicing in budding yeast.

RNA 2017  23: published online doi: 10.1261/rna.060830

Wallace, E. and Beggs, J.D.

RNA splicing, an essential part of eukaryotic pre-messenger RNA processing, can be simultaneous with transcription by RNA polymerase II. Here, we compare and review independent next-generation sequencing methods that jointly quantify transcription and splicing in budding yeast. For many yeast transcripts, splicing is fast, taking place within seconds of intron transcription, while polymerase is within a few dozens of nucleotides of the 3’ splice site. Ribosomal protein transcripts are spliced particularly fast and co-transcriptionally. However, some transcripts are spliced inefficiently or mainly post-transcriptionally. Intron-mediated regulation of some genes is likely to be co-transcriptional. We suggest that intermediates of the splicing reaction, missing from current datasets, may hold key information about splicing kinetics.


Transcriptome-wide RNA processing kinetics revealed using extremely short 4tU labeling.

Genome Biology 2015 16:282. PMC4699367.

Barrass JD, Reid JEA, Huang Y, Hector RD, Sanguinetti G, Beggs JD, Granneman S.

Background: RNA levels detected at steady state are the consequence of multiple dynamic processes within the cell. In addition to synthesis and decay, transcripts undergo processing. Metabolic tagging with a nucleotide analog is one way of determining the relative contributions of synthesis, decay and conversion processes globally.

Results: By improving 4-thiouracil labeling of RNA in Saccharomyces cerevisiae we were able to isolate RNA produced during as little as 1 minute, allowing the detection of nascent pervasive transcription. Nascent RNA labeled for 1.5, 2.5 or 5 minutes was isolated and analyzed by reverse transcriptase-quantitative polymerase chain reaction and RNA sequencing. High kinetic resolution enabled detection and analysis of short-lived non-coding RNAs as well as intron-containing pre-mRNAs in wild-type yeast. From these data we measured the relative stability of pre-mRNA species with different high turnover rates and investigated potential correlations with sequence features.

Conclusions: Our analysis of non-coding RNAs reveals a highly significant association between non-coding RNA stability, transcript length and predicted secondary structure. Our quantitative analysis of the kinetics of pre-mRNA splicing in yeast reveals that ribosomal protein transcripts are more efficiently spliced if they contain intron secondary structures that are predicted to be less stable. These data, in combination with previous results, indicate that there is an optimal range of stability of intron secondary structures that allows for rapid splicing.


Cwc21p promotes the second step conformation of the spliceosome and modulates 3' splice site selection

Nucl. Acids Res. 2015 43: 3309–3317. PMC4381068

Gautam, A., Grainger, R., Barrass, J.D. Vilardell, J. and Beggs, J.D.

Pre-mRNA splicing involves two transesterification steps catalyzed by the spliceosome. How RNA substrates are positioned in each step and the molecular rearrangements involved, remain obscure. Here, we show that mutations in PRP16, PRP8, SNU114 and the U5 snRNA that affect this process interact genetically with CWC21, that encodes the yeast orthologue of the human SR protein, Rm300/SRRM2. Our microarray analysis shows changes in 3_ splice site selection at elevated temperature in a subset of introns in cwc21Δ cells. Considering all the available data, we propose a role for Cwc21p positioning the 3_ splice site at the transition to the second step conformation of the spliceosome, mediated through its interactions with the U5 snRNP. This suggests a mechanism whereby SRm300/SRRM2, might influence splice site selection in human cells.


Brr2p carboxy-termianl Sec63 domain modulates Prp16 splicing helicase

Nucl. Acids Res. 2014 42:13897-910. PMC4267655.

Cordin O, Hahn D, Alexander RD, Gautam A, Saveanu C, Barrass JD, Beggs JD.

RNA helicases are essential for virtually all cellular processes, however, their regulation is poorly understood. The activities of eight RNA helicases are required for pre-mRNA splicing. Amongst these, Brr2p is unusual in having two helicase modules, of which only the amino-terminal helicase domain appears to be catalytically active. Using genetic and biochemical approaches, we investigated interaction of the carboxy-terminal helicase module, in particular the carboxy-terminal Sec63-2 domain, with the splicing RNA helicase Prp16p. Combining mutations in BRR2 and PRP16 suppresses or enhances physical interaction and growth defects in an allele-specific manner, signifying functional interactions. Notably, we show that Brr2p Sec63-2 domain can modulate the ATPase activity of Prp16p in vitro by interfering with its ability to bind RNA. We therefore propose that the carboxy-terminal helicase module of Brr2p acquired a regulatory function that allows Brr2p to modulate the ATPase activity of Prp16p in the spliceosome by controlling access to its RNA substrate/cofactor.


A splicing-dependent transcriptional checkpoint associated with prespliceosome formation

Mol Cell. 2014 Mar 6;53(5):779-90.
Chathoth KT, Barrass JD, Webb S, Beggs JD

There is good evidence for functional interactions between splicing and transcription in eukaryotes, but how and why these processes are coupled remain unknown. Prp5 protein (Prp5p) is an RNA-stimulated adenosine triphosphatase (ATPase) required for prespliceosome formation in yeast. We demonstrate through in vivo RNA labeling that, in addition to a splicing defect, the prp5-1 mutation causes a defect in the transcription of intron-containing genes. We present chromatin immunoprecipitation evidence for a transcriptional elongation defect in which RNA polymerase that is phosphorylated at Ser5 of the largest subunit's heptad repeat accumulates over introns and that this defect requires Cus2 protein. A similar accumulation of polymerase was observed when prespliceosome formation was blocked by a mutation in U2 snRNA. These results indicate the existence of a transcriptional elongation checkpoint that is associated with prespliceosome formation during cotranscriptional spliceosome assembly. We propose a role for Cus2p as a potential checkpoint factor in transcription


A rule-based kinetic model of RNA polymerase II C-terminal domain phosphorylation

J R Soc Interface. 2013 Jun 26;10(86):20130438

Aitken S, Alexander RD, Beggs JD.

The complexity of many RNA processing pathways is such that a conventional systems modelling approach is inadequate to represent all the molecular species involved. We demonstrate that rule-based modelling permits a detailed model of a complex RNA signalling pathway to be defined. Phosphorylation of the RNA polymerase II (RNAPII) C-terminal domain (CTD; a flexible tail-like extension of the largest subunit) couples pre-messenger RNA capping, splicing and 3' end maturation to transcriptional elongation and termination, and plays a central role in integrating these processes. The phosphorylation states of the serine residues of many heptapeptide repeats of the CTD alter along the coding region of genes as a function of distance from the promoter. From a mechanistic perspective, both the changes in phosphorylation and the location at which they take place on the genes are a function of the time spent by RNAPII in elongation as this interval provides the opportunity for the kinases and phosphatases to interact with the CTD. On this basis, we synthesize the available data to create a kinetic model of the action of the known kinases and phosphatases to resolve the phosphorylation pathways and their kinetics


Structural basis for dual roles of Aar2p in U5 snRNP assembly

Genes Dev. 2013 Mar 1;27(5):525-40.

Weber G, Cristão VF, Santos KF, Jovin SM, Heroven AC, Holton N, Lührmann R, Beggs JD, Wahl MC.

Yeast U5 small nuclear ribonucleoprotein particle (snRNP) is assembled via a cytoplasmic precursor that contains the U5-specific Prp8 protein but lacks the U5-specific Brr2 helicase. Instead, pre-U5 snRNP includes the Aar2 protein not found in mature U5 snRNP or spliceosomes. Aar2p and Brr2p bind competitively to a C-terminal region of Prp8p that comprises consecutive RNase H-like and Jab1/MPN-like domains. To elucidate the molecular basis for this competition, we determined the crystal structure of Aar2p in complex with the Prp8p RNase H and Jab1/MPN domains. Aar2p binds on one side of the RNase H domain and extends its C terminus to the other side, where the Jab1/MPN domain is docked onto a composite Aar2p-RNase H platform. Known Brr2p interaction sites of the Jab1/MPN domain remain available, suggesting that Aar2p-mediated compaction of the Prp8p domains sterically interferes with Brr2p binding. Moreover, Aar2p occupies known RNA-binding sites of the RNase H domain, and Aar2p interferes with binding of U4/U6 di-snRNA to the Prp8p C-terminal region. Structural and functional analyses of phospho-mimetic mutations reveal how phosphorylation reduces affinity of Aar2p for Prp8p and allows Brr2p and U4/U6 binding. Our results show how Aar2p regulates both protein and RNA binding to Prp8p during U5 snRNP assembly.


RNA helicases in splicing

RNA Biology 2013 Jan;10(1):83-95

Cordin, O. and Beggs, J.D.

In eukaryotic cells, introns are spliced from pre-mRNAs by the spliceosome. Both the composition and the structure of the spliceosome are highly dynamic, and eight DExD/H RNA helicases play essential roles in controlling conformational rearrangements. There is evidence that the various helicases are functionally and physically connected with each other and with many other factors in the spliceosome. Understanding the dynamics of those interactions is essential to comprehend the mechanism and regulation of normal as well as of pathological splicing. This review focuses on recent advances in the characterization of the splicing helicases and their interactions, and highlights the deep integration of splicing helicases in global mRNP biogenesis pathways.


Brr2-mediated conformational rearrangements in the spliceosome during spliceosome activation and substrate repositioning


Genes Dev. 2012 26:2408-2421.


Hahn, D., Kudla, G., Tollervey, D. and Beggs, J.D


Brr2p is one of eight RNA helicases involved in pre-mRNA splicing. Detailed understanding of the functions of Brr2p and other spliceosomal helicases has been limited by lack of knowledge of their in vivo substrates. To address this, sites of direct Brr2p-RNA interaction were identified by in vivo UV cross-linking in budding yeast. Cross-links identified in the U4 and U6 small nuclear RNAs (snRNAs) suggest U4/U6 stem I as a Brr2p substrate during spliceosome activation. Further Brr2p cross-links were identified in loop 1 of the U5 snRNA and near splice sites and 3' ends of introns, suggesting the possibility of a previously uncharacterized function for Brr2p in the catalytic center of the spliceosome. Consistent with this, mutant brr2-G858R reduced second-step splicing efficiency and enhanced cross-linking to 3' ends of introns. Furthermore, RNA sequencing indicated preferential inhibition of splicing of introns with structured 3' ends. The Brr2-G858Rp cross-linking pattern in U6 was consistent with an open conformation for the catalytic center of the spliceosome during first-to-second-step transition. We propose a previously unsuspected function for Brr2p in driving conformational rearrangements that lead to competence for the second step of splicing.


Kinetic analysis of pre-ribosome structure in vivo

RNA, 2012 18: PMID:23093724.

Swiatkowska, A., Wlotzka, W., Tuck, A., Barrass, J.D., Beggs, J.D. and Tollervey, D.

Pre-ribosomal particles undergo numerous structural changes during maturation, but their high complexity and short lifetimes make these changes very difficult to follow in vivo. In consequence, pre-ribosome structure and composition have largely been inferred from purified particles and analyzed in vitro. Here we describe techniques for kinetic analyses of the changes in pre-ribosome structure in living cells of Saccharomyces cerevisiae. To allow this, in vivo structure probing by DMS modification was combined with affinity purification of newly synthesized 20S pre-rRNA over a time course of metabolic labeling with 4-thiouracil. To demonstrate that this approach is generally applicable, we initially analyzed the accessibility of the region surrounding cleavage site D site at the 3' end of the mature 18S rRNA region of the pre-rRNA. This revealed a remarkably flexible structure throughout 40S subunit biogenesis, with little stable RNA-protein interaction apparent. Analysis of folding in the region of the 18S central pseudoknot was consistent with previous data showing U3 snoRNA-18S rRNA interactions. Dynamic changes in the structure of the hinge between helix 28 (H28) and H44 of pre-18S rRNA were consistent with recently reported interactions with the 3' guide region of U3 snoRNA. Finally, analysis of the H18 region indicates that the RNA structure matures early, but additional protection appears subsequently, presumably reflecting protein binding. The structural analyses described here were performed on total, affinity-purified, newly synthesized RNA, so many classes of RNA and RNA-protein complex are potentially amenable to this approach.


Structure, function and regulation of spliceosomal RNA helicases.

Curr. Opin. Cell Biol. 2012 24:431-438 PMID:22464735. Not Open Access

Cordin, O., Hahn, D. and Beggs, J.D.


Mechanism for Aar2p function as a U5 snRNP assembly factor

Genes Dev. 2011 Aug 1;25(15):1601-12

Weber G, Cristão VF, de L Alves F, Santos KF, Holton N, Rappsilber J, Beggs# JD, Wahl MC.
‡these authors contributed equally; #Corresponding author.

Little is known about how particle-specific proteins are assembled on spliceosomal small nuclear ribonucleoproteins (snRNPs). Brr2p is a U5 snRNP-specific RNA helicase required for spliceosome catalytic activation and disassembly. In yeast, the Aar2 protein is part of a cytoplasmic precursor U5 snRNP that lacks Brr2p and is replaced by Brr2p in the nucleus. Here we show that Aar2p and Brr2p bind to different domains in the C-terminal region of Prp8p; Aar2p interacts with the RNaseH domain, whereas Brr2p interacts with the Jab1/MPN domain. These domains are connected by a long, flexible linker, but the Aar2p-RNaseH complex sequesters the Jab1/MPN domain, thereby preventing binding by Brr2p. Aar2p is phosphorylated in vivo, and a phospho-mimetic S253E mutation in Aar2p leads to disruption of the Aar2p-Prp8p complex in favor of the Brr2p-Prp8p complex. We propose a model in which Aar2p acts as a phosphorylation-controlled U5 snRNP assembly factor that regulates the incorporation of the particle-specific Brr2p. The purpose of this regulation may be to safeguard against nonspecific RNA binding to Prp8p and/or premature activation of Brr2p activity.


Cross-linking, ligation, and sequencing of hybrids reveals RNA-RNA interactions in yeast.

Proc Natl Acad Sci U S A. 2011 Jun 14;108(24):10010-5.

Kudla G, Granneman S, Hahn D, Beggs JD, Tollervey D.
‡these authors contributed equally

Many protein-protein and protein-nucleic acid interactions have been experimentally characterized, whereas RNA-RNA interactions have generally only been predicted computationally. Here, we describe a high-throughput method to identify intramolecular and intermolecular RNA-RNA interactions experimentally by cross-linking, ligation, and sequencing of hybrids (CLASH). As validation, we identified 39 known target sites for box C/D modification-guide small nucleolar RNAs (snoRNAs) on the yeast pre-rRNA. Novel snoRNA-rRNA hybrids were recovered between snR4-5S and U14-25S. These are supported by native electrophoresis and consistent with previously unexplained data. The U3 snoRNA was found to be associated with sequences close to the 3' side of the central pseudoknot in 18S rRNA, supporting a role in formation of this structure. Applying CLASH to the yeast U2 spliceosomal snRNA led to a revised predicted secondary structure, featuring alternative folding of the 3' domain and long-range contacts between the 3' and 5' domains. CLASH should allow transcriptome-wide analyses of RNA-RNA interactions in many organisms.

Splicing-dependent RNA polymerase pausing in yeast.

Faculty of 1000 Rating 8 - Must Read

Mol Cell. 2010 Nov 24;40(4):582-93.

Alexander RD, Innocente SA, Barrass JD, Beggs JD.

In eukaryotic cells, there is evidence for functional coupling between transcription and processing of pre-mRNAs. To better understand this coupling, we performed a high-resolution kinetic analysis of transcription and splicing in budding yeast. This revealed that shortly after induction of transcription, RNA polymerase accumulates transiently around the 3' end of the intron on two reporter genes. This apparent transcriptional pause coincides with splicing factor recruitment and with the first detection of spliced mRNA and is repeated periodically thereafter. Pausing requires productive splicing, as it is lost upon mutation of the intron and restored by suppressing the splicing defect. The carboxy-terminal domain of the paused polymerase large subunit is hyperphosphorylated on serine 5, and phosphorylation of serine 2 is first detected here. Phosphorylated polymerase also accumulates around the 3' splice sites of constitutively expressed, endogenous yeast genes. We propose that transcriptional pausing is imposed by a checkpoint associated with cotranscriptional splicing.


RiboSys, a high-resolution, quantitative approach to measure the in vivo kinetics of pre-mRNA splicing and 3'-end processing in Saccharomyces cerevisiae.

RNA. 2010 Dec;16(12):2570-80.

Alexander RD, Barrass JD, Dichtl B, Kos M, Obtulowicz T, Robert MC, Koper M, Karkusiewicz I, Mariconti L, Tollervey D, Dichtl B, Kufel J, Bertrand E, Beggs JD.

We describe methods for obtaining a quantitative description of RNA processing at high resolution in budding yeast. As a model gene expression system, we constructed tetON (for induction studies) and tetOFF (for repression, derepression, and RNA degradation studies) yeast strains with a series of reporter genes integrated in the genome under the control of a tetO7 promoter. Reverse transcription and quantitative real-time-PCR (RT-qPCR) methods were adapted to allow the determination of mRNA abundance as the average number of copies per cell in a population. Fluorescence in situ hybridization (FISH) measurements of transcript numbers in individual cells validated the RT-qPCR approach for the average copy-number determination despite the broad distribution of transcript levels within a population of cells. In addition, RT-qPCR was used to distinguish the products of the different steps in splicing of the reporter transcripts, and methods were developed to map and quantify 3'-end cleavage and polyadenylation. This system permits pre-mRNA production, splicing, 3'-end maturation and degradation to be quantitatively monitored with unprecedented kinetic detail, suitable for mathematical modeling. Using this approach, we demonstrate that reporter transcripts are spliced prior to their 3'-end cleavage and polyadenylation, that is, cotranscriptionally.


Cross-talk in transcription, splicing and chromatin: who makes the first call?

Biochem Soc Trans. 2010 Oct;38(5):1251-6.

Alexander R and Beggs JD.

The complex processes of mRNA transcription and splicing were traditionally studied in isolation. In vitro studies showed that splicing could occur independently of transcription and the perceived wisdom was that, to a large extent, it probably did. However, there is now abundant evidence for functional interactions between transcription and splicing, with important consequences for splicing regulation. In the present paper, we summarize the evidence that transcription affects splicing and vice versa, and the more recent indications of epigenetic effects on splicing, through chromatin modifications. We end by discussing the potential for a systems biology approach to obtain better insight into how these processes affect each other.

Brr2p RNA helicase with a split personality: insights into structure and function.

Biochem Soc Trans. 2010 Aug;38(4):1105-9.

Hahn D and Beggs JD.

RNA helicases are involved in many cellular processes. Pre-mRNA splicing requires eight different DExD/H-box RNA helicases, which facilitate spliceosome assembly and remodelling of the intricate network of RNA rearrangements that are central to the splicing process. Brr2p, one of the spliceosomal RNA helicases, stands out through its unusual domain architecture. In the present review we highlight the advances made by recent structural and biochemical studies that have important implications for the mechanism and regulation of Brr2p activity. We also discuss the involvement of human Brr2 in retinitis pigmentosa, a degenerative eye disease, and how its functions in splicing might connect to the molecular pathology of the disease.


Prognosis for splicing factor PRPF8 retinitis pigmentosa, novel mutations and correlation between human and yeast phenotypes.

Hum Mutat. 2010 May;31(5):E1361-76.

Towns KV, Kipioti A, Long V, McKibbin M, Maubaret C, Vaclavik V, Ehsani P, Springell K, Kamal M, Ramesar RS, Mackey DA, Moore AT, Mukhopadhyay R, Webster AR, Black GC, O'Sullivan J, Bhattacharya SS, Pierce EA, Beggs JD, Inglehearn CF.

PRPF8-retinitis pigmentosa is said to be severe but there has been no overview of phenotype across different mutations. We screened RP patients for PRPF8 mutations and identified three new missense mutations, including the first documented mutation outside exon 42 and the first de novo mutation. This brings the known RP-causing mutations in PRPF8 to nineteen. We then collated clinical data from new and published cases to determine an accurate prognosis for PRPF8-RP. Clinical data for 75 PRPF8-RP patients were compared, revealing that while the effect on peripheral retinal function is severe, patients generally retain good visual acuity in at least one eye until the fifth or sixth decade. We also noted that prognosis for PRPF8-RP differs with different mutations, with p.H2309P or p.H2309R having a worse prognosis than p.R2310K. This correlates with the observed difference in growth defect severity in yeast lines carrying the equivalent mutations, though such correlation remains tentative given the limited number of mutations for which information is available. The yeast phenotype is caused by lack of mature spliceosomes in the nucleus, leading to reduced RNA splicing function. Correlation between yeast and human phenotypes suggests that splicing factor RP may also result from an underlying splicing deficit.

Processivity and coupling in messenger RNA transcription.

PLoS One. 2010 Jan 28;5(1):e8845.

Aitken S, Robert MC, Alexander RD, Goryanin I, Bertrand E, Beggs JD.

The complexity of messenger RNA processing is now being uncovered by experimental techniques that are capable of detecting individual copies of mRNA in cells, and by quantitative real-time observations that reveal the kinetics. This processing is commonly modelled by permitting mRNA to be transcribed only when the promoter is in the on state. In this simple on/off model, the many processes involved in active transcription are represented by a single reaction. These processes include elongation, which has a minimum time for completion and processing that is not captured in the model.
In this paper, we explore the impact on the mRNA distribution of representing the elongation process in more detail. Consideration of the mechanisms of elongation leads to two alternative models of the coupling between the elongating polymerase and the state of the promoter: Processivity allows polymerases to complete elongation irrespective of the promoter state, whereas coupling requires the promoter to be active to produce a full-length transcript. We demonstrate that these alternatives have a significant impact on the predicted distributions. Models are simulated by the Gillespie algorithm, and the third and fourth moments of the resulting distribution are computed in order to characterise the length of the tail, and sharpness of the peak. By this methodology, we show that the moments provide a concise summary of the distribution, showing statistically-significant differences across much of the feasible parameter range.
We conclude that processivity is not fully consistent with the on/off model unless the probability of successfully completing elongation is low--as has been observed. The results also suggest that some form of coupling between the promoter and a rate-limiting step in transcription may explain the cell's inability to maintain high mRNA levels at low noise--a prediction of the on/off model that has no supporting evidence.

Physical and genetic interactions of yeast Cwc21p, an ortholog of human SRm300/SRRM2, suggest a role at the catalytic center of the spliceosome.

RNA. 2009 15(12):2161-73.

Grainger RJ, Barrass JD, Jacquier A, Rain JC, Beggs JD.

In Saccharomyces cerevisiae, Cwc21p is a protein of unknown function that is associated with the NineTeen Complex (NTC), a group of proteins involved in activating the spliceosome to promote the pre-mRNA splicing reaction. Here, we show that Cwc21p binds directly to two key splicing factors-namely, Prp8p and Snu114p-and becomes the first NTC-related protein known to dock directly to U5 snRNP proteins. Using a combination of proteomic techniques we show that the N-terminus of Prp8p contains an intramolecular fold that is a Snu114p and Cwc21p interacting domain (SCwid). Cwc21p also binds directly to the C-terminus of Snu114p. Complementary chemical cross-linking experiments reveal reciprocal protein footprints between the interacting Prp8 and Cwc21 proteins, identifying the conserved cwf21 domain in Cwc21p as a Prp8p binding site. Genetic and functional interactions between Cwc21p and Isy1p indicate that they have related functions at or prior to the first catalytic step of splicing, and suggest that Cwc21p functions at the catalytic center of the spliceosome, possibly in response to environmental or metabolic changes. We demonstrate that SRm300, the only SR-related protein known to be at the core of human catalytic spliceosomes, is a functional ortholog of Cwc21p, also interacting directly with Prp8p and Snu114p. Thus, the function of Cwc21p is likely conserved from yeast to humans.


Mutations in the U5 snRNA result in a non-uniform splicing deficiency and a reduction of Prp8p.

RNA 2009 15:1292-304.

Kershaw, C.J., Barrass, J.D., Beggs#, J.D., and O'Keefe#, R.T.
# Corresponding authors.

The U5 snRNA loop 1 aligns the 5? and 3? exons for ligation during the second step of pre-mRNA splicing. U5 is intimately associated with Prp8, which mediates pre-mRNA repositioning within the catalytic core of the spliceosome and interacts directly with U5 loop 1. The genome-wide effect of three U5 loop 1 mutants has been assessed by microarray analysis. These mutants exhibited impaired and improved splicing of subsets of pre-mRNAs compared to wild-type U5. Analysis of pre-mRNAs that accumulate revealed a change in base prevalence at specific positions near the splice sites. Analysis of processed pre-mRNAs exhibiting mRNA accumulation revealed a bias in base prevalence at one position within the 5? exon. While U5 loop 1 can interact with some of these positions the base bias is not directly related to sequence changes in loop 1. All positions that display a bias in base prevalence are at or next to positions known to interact with Prp8. Analysis of Prp8 in the presence of the three U5 loop 1 mutants revealed that the most severe mutant displayed reduced Prp8 stability. Depletion of U5 snRNA in vivo also resulted in reduced Prp8 stability. Our data suggest that certain mutations in U5 loop 1 perturb the stability of Prp8 and may affect interactions of Prp8 with a subset of pre-mRNAs influencing their splicing. Therefore, the integrity of U5 is important for the stability of Prp8 during splicing and provides one possible explanation for why U5 loop 1 and Prp8 are so highly conserved.

Requirements for nuclear localization of Lsm2-8p and competition between nuclear and cytoplasmic Lsm complexes

J. Cell Sci. (2007) 120: 4310-20

Michael P. Spiller, Martin A.M. Reijns and Jean D. Beggs

Lsm proteins are ubiquitous, multifunctional proteins that are involved in the processing and/or turnover of many RNAs. In eukaryotes, a hetero-heptameric complex of Lsm proteins (Lsm2-8p) affects the processing of small stable RNAs and pre-mRNAs in the nucleus, while a different hetero-heptameric complex of Lsm proteins (Lsm1-7p) promotes mRNA decapping and decay in the cytoplasm. These two complexes have six constituent proteins in common, yet localize to separate cellular compartments and perform apparently disparate functions. Little is known about the biogenesis of the Lsm complexes, or how they are recruited to different cellular compartments. We show that in yeast, the nuclear accumulation of/ /Lsm proteins depends on complex formation and that the Lsm8p subunit plays a crucial role. The nuclear localization of Lsm8p is itself most strongly influenced by Lsm2p and Lsm4p, its presumed neighbors in the Lsm2-8p complex. Furthermore, over-expression and depletion experiments imply that Lsm1p and Lsm8p act competitively with respect to the localization of the two complexes, suggesting a potential mechanism for co-regulation of nuclear and cytoplasmic RNA processing. A shift of Lsm proteins from the nucleus to the cytoplasm under stress conditions indicates that this competition is biologically significant.

Prp8 mutations that cause human retinitis pigmentosa lead to a U5 snRNP maturation defect in yeast

Nature Struc. Mol. Biol (2007) 14: 1077 - 1083; doi: 10.1038/nsmb1303 (Faculty of 1000 Recommended Reading)

Kum-Loong Boon, Richard J Grainger, Parastoo Ehsani, J. David Barrass, Tatsiana Auchynnikava, Chris F Inglehearn and Jean D. Beggs

Prp8 protein is a highly conserved pre-mRNA splicing factor and a component of spliceosomal U5 snRNPs. Intriguingly, although it is ubiquitously expressed, mutations in the C-terminus of human Prp8p cause the retina-specific disease Retinitis pigmentosa (RP). The biogenesis of U5 snRNPs is poorly characterised. We present evidence for a cytoplasmic precursor U5 snRNP in yeast that lacks a mature U5 snRNP component, Brr2p, and depends on a nuclear localization signal in Prp8p for its efficient nuclear import. The association of Brr2p with the U5 snRNP occurs within the nucleus. RP mutations in Prp8p in yeast result in nuclear accumulation of the precursor U5 snRNP, apparently as a consequence of disrupting the interaction of Prp8p with Brr2p. We therefore propose a novel assembly pathway for U5 snRNP complexes, which is disrupted by mutations that cause human RP.

The Lsm2-8 complex determines nuclear localization of the spliceosomal U6 snRNA

Nucleic Acids Research (2007) 35: 923-929; doi: 10.1093/nar/gkl1130

Michael P. Spiller, Kum-Loong Boon, Martin A. M. Reijns and Jean D. Beggs

Lsm proteins are ubiquitous, multifunctional proteins that are involved in the processing and/or turnover of many, if not all, RNAs in eukaryotes. They generally interact only transiently with their substrate RNAs, in keeping with their likely roles as RNA chaperones. The spliceosomal U6 snRNA is an exception, being stably associated with the Lsm2-8 complex. The U6 snRNA is generally considered to be intrinsically nuclear but the mechanism of its nuclear retention has not been demonstrated, although La protein has been implicated. We show here that the complete Lsm2-8 complex is required for nuclear accumulation of U6 snRNA in yeast. Therefore, just as Sm proteins effect nuclear localization of the other spliceosomal snRNPs, the Lsm proteins mediate U6 snRNP localization except that nuclear retention is the likely mechanism for the U6 snRNP. La protein, which binds only transiently to the nascent U6 transcript, has a smaller, apparently indirect, effect on U6 localization that is compatible with its proposed role as a chaperone in facilitating U6 snRNP assembly.

Yeast Ntr1/Spp382 mediates Prp43 function in post-spliceosomes

Molecular and Cellular Biology (2006) 26: 6016-6023

Kum-Loong Boon, Tatsiana Auchynnikava, Gretchen Edwalds-Gilbert, J. David Barrass, Alastair P. Droop, Christophe Dez and Jean D. Beggs

The Ntr1 and Ntr2 proteins of Saccharomyces cerevisiae have been reported to interact with proteins involved in pre-mRNA splicing but their roles in the splicing process are unknown. We show here that they associate with a post-splicing complex containing the excised intron and the spliceosomal U2, U5, and U6 snRNAs, supporting a link with a late stage in the pre-mRNA splicing process. Extract from cells that had been metabolically depleted of Ntr1 has low splicing activity and accumulates the excised intron. Also, the level of U4/U6 di-snRNP is increased, but the free U5 and U6 snRNPs are decreased in Ntr1-depleted extract, and increased levels of U2 and decreased levels of U4 are found associated with the U5 snRNP protein, Prp8. These results suggest a requirement for Ntr1 for turnover of the excised intron complex and recycling of snRNPs. Ntr1 interacts directly or indirectly with the intron release factor, Prp43, and is required for its association with the excised intron. We propose that Ntr1 promotes release of excised introns from splicing complexes by acting as a spliceosome receptor or RNA targeting factor for Prp43, possibly assisted by Ntr2 protein.


Prp8p dissection reveals domain structure and protein interaction sites

RNA (2006) 12: 198-205

Kum-Loong Boon, Christine M. Norman, Richard J. Grainger, Andrew J Newman and Jean D. Beggs

We describe a novel approach to characterize the functional domains of a protein in vivo. This involves the use of a custom-built Tn5-based transposon that causes the expression of a target gene as two contiguous polypeptides. When used as a genetic screen to dissect the budding yeast PRP8 gene, this showed that Prp8 protein could be dissected into three distinct pairs of functional polypeptides. Thus, four functional domains can be defined in the 2413-residue Prp8 protein, with boundaries in the regions of amino acids 394-443, 770, and 2170-2179. The central region of the protein was resistant to dissection by this approach, suggesting that it represents one large functional unit. The dissected constructs allowed investigation of factors that associate strongly with the N-or the C-terminal Prp8 protein fragments. Thus, the U5 snRNP protein Snu114p associates with Prp8p in the region 437-770, whereas fragmenting Prp8p at residue 2173 destabilizes its association with Aar2p.


Lsm proteins and RNA processing

Novartis Medal Lecture - Biochemical Society Transactions (2005) 33; 433-438

Jean Beggs

Sm and Lsm proteins are ubiquitous in eukaryotes and form complexes that interact with RNAs involved in almost every cellular process. My laboratory has studied the Lsm proteins in the yeast Saccharomyces cerevisiae, identifying in the nucleus and cytoplasm distinct complexes that affect pre-mRNA splicing and degradation, small nucleolar RNA, tRNA processing, rRNA processing and mRNA degradation. These activities suggest RNA chaperone-like roles for Lsm proteins, affecting RNA-RNA and/or RNA protein interactions. This article reviews the properties of the Sm and Lsm proteins and structurally and functionally related proteins in archaea and eubacteria.



Crosstalk between RNA metabolic pathways: an RNOMICS approach


Jean D. Beggs and David Tollervey

Eukaryotic cells contain many different RNA species. The nuclear pre-mRNAs and cytoplasmic mRNAs carry genomic information to the protein synthesis machinery, while many stable RNA species play major functional roles. The mature, functional forms of all of these RNA species are generated by post-transcriptional processing and evidence has been accumulating for functional links between the various processing pathways. This suggests the existence of regulatory networks that coordinate different stages of RNA metabolism. This article describes the aims and results to date of an EC-funded RNOMICS project as an example of an integrated approach to investigate these links.


Prp8 Protein; at the Heart of the Spliceosome

RNA (2005) 11: 533-557

Richard J. Grainger and Jean D. Beggs

Pre-messenger RNA (pre-mRNA) splicing is a central step in gene expression. Lying between transcription and protein synthesis, pre-mRNA splicing removes sequences (introns) that would otherwise disrupt the coding potential of intron-containing transcripts. This process takes place in the nucleus, catalysed by a large RNA-protein complex called the spliceosome. Prp8p, one of the largest and most highly conserved of nuclear proteins, occupies a central position in the catalytic core of the spliceosome, and has been implicated in several crucial molecular rearrangements that occur there. Recently, Prp8p has also come under the spotlight for its role in the inherited human disease, Retinitis Pigmentosa. Prp8 is unique, having no obvious homology to other proteins, however, using bioinformatical analysis we reveal the presence of a conserved RNA recognition motif (RRM), an MPN/JAB domain and a putative nuclear localization signal (NLS). Here, we review biochemical and genetical data, mostly related to the human and yeast proteins, that describe Prp8's central role within the spliceosome and its molecular interactions during spliceosome formation, as splicing proceeds, and in post-splicing complexes.


Interaction between a G-Patch Protein and a Spliceosomal DEXD/H-Box ATPase That Is Critical for Splicing

Molecular and Cellular Biology (2004) 24: 10101-10110

Edward J. Silverman, Ayaka Maeda, Janet Wei, Paul Smith, Jean D. Beggs, and Ren-Jang Lin,

Prp2 is an RNA-dependent ATPase that activates the spliceosome before the first transesterification reaction of pre-mRNA splicing. Prp2 has extensive homology throughout the helicase domain characteristic of DEXD/ H-box helicases and a conserved carboxyl-terminal domain also found in the spliceosomal helicases Prp16, Prp22, and Prp43. Despite the extensive homology shared by these helicases, each has a distinct, sequential role in splicing; thus, uncovering the determinants of specificity becomes crucial to the understanding of Prp2 and the other DEAH-splicing helicases. Mutations in an 11-mer near the C-terminal end of Prp2 eliminate its spliceosome binding and splicing activity. Here we show that a helicase-associated protein interacts with this domain and that this interaction contributes to the splicing process. First, a genome-wide yeast two-hybrid screen using Prp2 as bait identified Spp2, which contained a motif with glycine residues found in a number of RNA binding proteins. SPP2 was originally isolated as a genetic suppressor of a prp2 mutant. In a reciprocal screen, Spp2 specifically pulled out the C-terminal half of Prp2. Mutations in the Prp2 C-terminal 11-mer that disrupted function or spliceosome binding also disrupted Spp2 interaction. A screen of randomly mutagenized SPP2 clones identified an Spp2 protein with a mutation in the G patch that could restore interaction with Prp2 and enhanced splicing in a prp2 mutant strain. The study identifies a potential mechanism for Prp2 specificity mediated through a unique interaction with Spp2 and elucidates a role for a helicase-associated protein in the binding of a DEXD/H-box protein to the spliceosome.


Nuclear Pre-mRNA Decapping and 5_ Degradation in Yeast Require the Lsm2-8p Complex

Molecular and Cellular Biology (2004) 24: 9646-9657

Joanna Kufel, Cecile Bousquet-Antonelli, Jean D. Beggs, and David Tollervey

Previous analyses have identified related cytoplasmic Lsm1-7p and nuclear Lsm2-8p complexes. Here we report that mature heat shock and MET mRNAs that are trapped in the nucleus due to a block in mRNA export were strongly stabilized in strains lacking Lsm6p or the nucleus-specific Lsm8p protein but not by the absence of the cytoplasmic Lsm1p. These nucleus-restricted mRNAs remain polyadenylated until their degradation, indicating that nuclear mRNA degradation does not involve the incremental deadenylation that is a key feature of cytoplasmic turnover. Lsm8p can be UV cross-linked to nuclear poly(A)_ RNA, indicating that an Lsm2-8p complex interacts directly with nucleus-restricted mRNA. Analysis of pre-mRNAs that contain intronic snoRNAs indicates that their 5_ degradation is specifically inhibited in strains lacking any of the Lsm2-8p proteins but Lsm1p. Nucleus-restricted mRNAs and pre-mRNA degradation intermediates that accumulate in lsm mutants remain 5_ capped. We conclude that the Lsm2-8p complex normally targets nuclear RNA substrates for decapping.


Lsm proteins promote regeneration of pre-mRNA splicing activity.

Current Biology (2004) 14: 1487-1491

Loredana Verdone#, Silvia Galardi#, David Page and Jean D. Beggs. #These authors contributed equally to this work

Lsm proteins are ubiquitous, multifunctional proteins that affect the processing of most RNAs in eukaryotic cells, but their function is unknown. A complex of seven Lsm proteins, Lsm2-8, associates with the U6 small nuclear RNA (snRNA) that is a component of spliceosome complexes in which pre-mRNA splicing occurs. Spliceosomes contain five snRNAs, U1, U2, U4, U5, and U6, that are packaged as ribonucleoprotein particles (snRNPs). U4 and U6 snRNAs contain extensive sequence complementarity and interact to form U4/U6 di-snRNPs. U4/U6 di-snRNPs associate with U5 snRNPs to form U4/U6.U5 tri-snRNPs prior to spliceosome assembly. Within spliceosomes, disruption of base-paired U4/U6 heterodimer allows U6 snRNA to form part of the catalytic center. Following completion of the splicing reaction, snRNPs must be recycled for subsequent rounds of splicing, although little is known about this process. Here we present evidence that regeneration of splicing activity in vitro is dependent on Lsm proteins. RNP reconstitution experiments with exogenous U6 RNA show that Lsm proteins promote the formation of U6-containing complexes and suggest that Lsm proteins have a chaperone-like function, supporting the assembly or remodeling of RNP complexes involved in splicing. Such a function could explain the involvement of Lsm proteins in a wide variety of RNA processing pathways.


A complex pathway for 3'processing of the yeast U3 snoRNA

Nucleic Acids Res. (2003)31(23):6788-97

Joanna Kufel, Christine Allmang, Loredana Verdone, Jean Beggs and David Tollervey

Mature U3 snoRNA in yeast is generated from the 3'-extended precursors by endonucleolytic cleavage followed by exonucleolytic trimming. These precursors terminate in poly(U) tracts and are normally stabilised by binding of the yeast La homologue, Lhp1p. We report that normal 3' processing of U3 requires the nuclear Lsm proteins. On depletion of any of the five essential proteins, Lsm2-5p or Lsm8p, the normal 3'-extended precursors to the U3 snoRNA were lost. Truncated fragments of both mature and pre-U3 accumulated in the Lsm-depleted strains, consistent with substantial RNA degradation. Pre-U3 species were co-precipitated with TAP-tagged Lsm3p, but the association with spliced pre-U3 was lost in strains lacking Lhp1p. The association of Lhp1p with pre-U3 was also reduced on depletion of Lsm3p or Lsm5p, indicating that binding of Lhp1p and the Lsm proteins is interdependent. In contrast, a tagged Sm-protein detectably co-precipitated spliced pre-U3 species only in strains lacking Lhp1p. We propose that the Lsm2-8p complex functions as a chaperone in conjunction with Lhp1p to stabilise pre-U3 RNA species during 3' processing. The Sm complex may function as a back-up to stabilise 3' ends that are not protected by Lhp1p.


Splicing goes global

TIG 9: 295-298 (2003)

J. David Barrass and Jean D. Beggs

Transcriptomics, the analysis of the complement of mRNAs transcribed from a cell’s genome, currently focuses mainly on mature, processed mRNAs. However, posttranscriptional processing of primary transcripts can significantly affect both the quantity and the structure of the mature mRNAs and therefore of the protein products. Recently, the development of an intronspecific microarray has permitted a preliminary analysis of the splicing of all intron-containing transcripts in Saccharomyces cerevisiae. Here, we discuss the findings and what might be learned from this kind of approach.


3'-processing of yeast tRNATrp precedes 5'-processing.

RNA 9(2): 202-8 (2003)

Kufel J, Tollervey D.

Previous analyses of eukaryotic pre-tRNAs processing have reported that 5'-cleavage by RNase P precedes 3'-maturation. Here we report that in contrast to all other yeast tRNAs analyzed to date, tRNA(Trp) undergoes 3'-maturation prior to 5'-cleavage. Despite its unusual processing pathway, pre-tRNA(Trp) resembles other pre-tRNAs, showing dependence on the essential Lsm proteins for normal processing and efficient association with the yeast La homolog, Lhp1p. tRNA(Trp) is also unusual in not requiring Lhp1p for 3' processing and stability. However, other Lhp1p-independent tRNAs, tRNA(2)(Lys) and tRNA(1)(Ile), follow the normal pathway of 5'-processing prior to 3-processing.


Identification and characterisation of Prp45p and Prp46p, essential pre-mRNA splicing factors.

RNA 9: 135-150 (2003)   

Michael Albers, Ann Diment, Mariela Muraru, Caroline S. Russell and Jean D. Beggs

Through exhaustive two-hybrid screens using a budding yeast genomic library, and starting with the splicing factor and DEAH-box RNA helicase Prp22p as bait, we identified yeast Prp45p and Prp46p. We show that as well as interacting in two-hybrid screens, Prp45p and Prp46p interact with each other in vitro. We demonstrate that Prp45p and Prp46p are spliceosome-associated throughout the splicing process and both are essential for pre-mRNA splicing. Under non-splicing conditions they also associate in coprecipitation assays with low levels of the U2, U5 and U6 snRNAs that may indicate their presence in endogenous activated spliceosomes or in a post-splicing snRNP complex.


LSM Proteins Are Required for Normal Processing and Stability of Ribosomal RNAs

J. Biol. Chem., Jan 2003; 278: 2147 - 2156

Joanna Kufel, Christine Allmang, Elisabeth Petfalski, Jean Beggs, and David Tollervey

Depletion of any of the essential Lsm proteins, Lsm2-5p or Lsm8p, delayed pre-rRNA processing and led to the accumulation of many aberrant processing intermediates, indicating that an Lsm complex is required to maintain the normally strict order of processing events. In addition, high levels of degradation products derived from both precursors and mature rRNAs accumulated in Lsm-depleted strains. Depletion of the essential Lsm proteins reduced the apparent processivity of both 5' and 3' exonuclease activities involved in 5.8S rRNA processing, and the degradation intermediates that accumulated were consistent with inefficient 5' and 3' degradation. Many, but not all, pre-rRNA species could be coprecipitated with tagged Lsm3p, but not with tagged Lsm1p or non-tagged control strains, suggesting their direct interaction with an Lsm2-8p complex. We propose that Lsm proteins facilitate RNA protein interactions and structural changes required during ribosomal subunit assembly.


Lsm Proteins Are Required for Normal Processing of Pre-tRNAs and Their Efficient Association with La-Homologous Protein Lhp1p

Mol. Cell. Biol. 22: 5248-5256. (2002)

Joanna Kufel, Christine Allmang, Loredana Verdone, Jean D. Beggs, and David Tollervey

Depletion of any of the five essential proteins Lsm2p to Lsm5p and Lsm8p leads to strong accumulation of all tested unspliced pre-tRNA species, as well as accumulation of 5' and 3' unprocessed species. Aberrant 3'-extended pre-tRNAs were detected, presumably due to stabilization of transcripts that fail to undergo correct transcription termination, and the accumulation of truncated tRNA fragments was also observed. Tandem affinity purification-tagged Lsm3p was associated with pre-tRNA primary transcripts and, less efficiently, with other unspliced pre-tRNA intermediates but not mature tRNAs. Association of the Saccharomyces cerevisiae La homologue Lhp1p with pre-tRNAs was reduced approximately threefold on depletion of Lsm3p or Lsm5p. The association of Lhp1p with larger RNA polymerase III transcripts, pre-RNase P RNA and the signal recognition particle RNA (scR1), was more drastically reduced. The impaired pre-tRNA processing seen on Lsm depletion is not, however, due solely to reduced Lhp1p association as evidenced by analysis of lhp1- strains depleted of Lsm3p or Lsm5p. These data are consistent with roles for an Lsm complex as a chaperone that facilitates the efficient association of pre-tRNA processing factors with their substrates.


Functional Contacts With a Range of Splicing Proteins Suggest a Central Role for Brr2p in the Dynamic Control of the Order of Events in Spliceosomes of Saccharomyces cerevisiae

Genetics 157:1451-1467 (2001)

Rob W. van Nues and Jean D. Beggs

Mapping of functional protein interactions will help in understanding conformational rearrangements that occur within large complexes like spliceosomes. Because the U5 snRNP plays a central role in pre-mRNA splicing, we undertook exhaustive two-hybrid screening with Brr2p, Prp8p, and other U5 snRNP-associated proteins. DExH-box protein Brr2p interacted specifically with five splicing factors: Prp8p, DEAH-box protein Prp16p, U1 snRNP protein Snp1p, second-step factor Slu7p, and U4/U6.U5 tri-snRNP protein Snu66p, which is required for splicing at low temperatures. Co-immunoprecipitation experiments confirmed direct or indirect interactions of Prp16p, Prp8p, Snu66p, and Snp1p with Brr2p and led us to propose that Brr2p mediates the recruitment of Prp16p to the spliceosome. We provide evidence that the prp8-1 allele disrupts an interaction with Brr2p, and we propose that Prp8p modulates U4/U6 snRNA duplex unwinding through another interaction with Brr2p. The interactions of Brr2p with a wide range of proteins suggest a particular function for the C-terminal half, bringing forward the hypothesis that, apart from U4/U6 duplex unwinding, Brr2p promotes other RNA rearrangements, acting synergistically with other spliceosomal proteins, including the structurally related Prp2p and Prp16p. Overall, these protein interaction studies shed light on how splicing factors regulate the order of events in the large spliceosome complex. [Full text]


Genetic and Physical Interactions Between Factors Involved in Both Cell Cycle Progression and Pre-mRNA Splicing in Saccharomyces cerevisiae

Genetics 156:1503-1517 (2000)

Sigal Ben-Yehuda, Ian Dix, Caroline S. Russell, Margaret McGarvey, Jean D. Beggs, and Martin Kupiec

The PRP17/CDC40 gene of Saccharomyces cerevisiae functions in two different cellular processes: pre-mRNA splicing and cell cycle progression. The Prp17/Cdc40 protein participates in the second step of the splicing reaction and, in addition, prp17/cdc40 mutant cells held at the restrictive temperature arrest in the G2 phase of the cell cycle. Here we describe the identification of nine genes that, when mutated, show synthetic lethality with the prp17/cdc40 allele. Six of these encode known splicing factors: Prp8p, Slu7p, Prp16p, Prp22p, Slt11p, and U2 snRNA. The other three, SYF1, SYF2, and SYF3, represent genes also involved in cell cycle progression and in pre-mRNA splicing. Syf1p and Syf3p are highly conserved proteins containing several copies of a repeated motif, which we term RTPR. This newly defined motif is shared by proteins involved in RNA processing and represents a subfamily of the known TPR (tetratricopeptide repeat) motif. Using two-hybrid interaction screens and biochemical analysis, we show that the SYF gene products interact with each other and with four other proteins: Isy1p, Cef1p, Prp22p, and Ntc20p. We discuss the role played by these proteins in splicing and cell cycle progression.


Functional analyses of interacting factors involved in both pre-mRNA splicing and cell cycle progression in Saccharomyces cerevisiae.


RNA 6:1565-72 (2000)

Caroline S. Russell, Sigal Ben-Yehuda,Ian Dix, Martin Kupiec,Jean D. Beggs

Through a genetic screen to search for factors that interact with Prp17/Cdc40p, a protein involved in both cell cycle progression and pre-mRNA splicing, we identify three novel factors, which we call Syf1p, Syf2p, and Syf3 (SYnthetic lethal with cdc Forty). Here we present evidence that all three proteins are spliceosome associated, that they associate weakly or transiently with U6 and U5 snRNAs, and that Syf1p and Syf3p (also known as Clf1p) are required for pre-mRNA splicing. In addition we show that depletion of Syf1p or Syf3p results in cell cycle arrest at the G2/M transition. Thus, like Prp17/Cdc40p, Syf1p and Syf3p are involved in two distinct cellular processes. We discuss the likelihood that Syf1p, Syf2p, and Syf3p are components of a protein complex that assembles into spliceosomes and also regulates cell cycle progression. [Full text]


Dhr1p, a Putative DEAH-Box RNA Helicase, Is Associated with the Box C+D snoRNP U3

Mol Cell Biol 20:7238-7246 (2000)

Alan Colley, Jean D. Beggs, David Tollervey, Denis L. Lafontaine

Putative RNA helicases are involved in most aspects of gene expression. All previously characterized members of the DEAH-box family of putative RNA helicases are involved in pre-mRNA splicing. Here we report the analysis of two novel DEAH-box RNA helicases, Dhr1p and Dhr2p, that were found to be predominantly nucleolar. Both genes are essential for viability, and MET-regulated alleles were therefore created. Depletion of Dhr1p or Dhr2p had no detectable effect on pre-mRNA splicing in vivo or vitro. Both Dhr1p and Dhr2p were, however, required for 18S rRNA synthesis. Depletion of Dhr2p inhibited pre-rRNA cleavage at sites A(0), A(1), and A(2), while Dhr1p depletion inhibited cleavage at sites A(1) and A(2). No coprecipitation of snoRNAs was detected with ProtA-Dhr2p, but Dhr1p-ProtA was stably associated with the U3 snoRNA. Depletion of Dhr1p inhibited processing steps that require base pairing of U3 to the 5' end of the 18S rRNA. We speculate that Dhr1p is targeted to the preribosomal particles by the U3-18S rRNA interaction and is required for the structural reorganization of the rRNA during formation of the central pseudoknot. [Full Text]


Genome-wide Protein Interaction Screens reveal Functional Networks Involving Sm-like Proteins


Yeast 17:95-110 (2000)

Micheline Fromont-Racine, Andrew E. Mayes, Adeline Brunet-Simon, Jean-Christophe Rain, Alan Colley, Ian Dix, Laurence Decourty, Nicolas Joly, Florence Ricard, Jean D. Beggs, Pierre Legrain

A set of seven structurally related Sm proteins forms the core of the snRNP particles containing the spliceosomal U1, U2, U4 and U5 snRNAs. A search of the genomic sequence of Saccharomyces cerevisiae has identified a number of open reading frames that potentially encode structurally similar proteins termed Lsm (Like Sm) proteins. With the aim of analysing all possible interactions between the Lsm proteins and any protein encoded in the yeast genome, we performed exhaustive and iterative genomic two-hybrid screens, starting with the Lsm proteins as baits. Indeed, extensive interactions amongst eight Lsm proteins were found that suggest the existence of a Lsm complex or complexes. These Lsm interactions apparently involve the conserved Sm domain that also mediates interactions between the Sm proteins. The screens also reveal functionally significant interactions with splicing factors, in particular with Prp4 and Prp24, compatible with genetic studies and with the reported association of Lsm proteins with spliceosomal U6 and U4/U6 particles. In addition, interactions with proteins involved in mRNA turnover, such as Mrt1, Dcp1, Dcp2 and Xrn1, point to roles for Lsm complexes in distinct RNA metabolic processes, that are confirmed in independent functional studies. These results provide compelling evidence that two-hybrid screens yield functionally meaningful information about protein-protein interactions and can suggest functions for uncharacterized proteins, especially when they are performed on a genome-wide scale


Yeast Sm-like proteins function in mRNA decapping and decay

Nature 404:515 - 518 (2000)

Sundaresan Tharun, Weihai He, Andew E. Mayes, Pascal Lennertz, Jean D. Beggs and Roy Parker

One of the main mechanisms of messenger RNA degradation in eukaryotes occurs by deadenylation-dependent decapping which leads to 5'-to-3' decay. A family of Sm-like (Lsm) proteins has been identified, members of which contain the 'Sm' sequence motif, form a complex with U6 small nuclear RNA and are required for pre-mRNA splicing. Here we show that mutations in seven yeast Lsm proteins (Lsm1-Lsm7) also lead to inhibition of mRNA decapping. In addition, the Lsm1-Lsm7 proteins co-immunoprecipitate with the mRNA decapping enzyme (Dcp1), a decapping activator (Pat1/Mrt1) and with mRNA. This indicates that the Lsm proteins may promote decapping by interactions with the mRNA and the decapping machinery. In addition, the Lsm complex that functions in mRNA decay appears to be distinct from the U6-associated Lsm complex, indicating that Lsm proteins form specific complexes that affect different aspects of mRNA metabolism. 


Extensive genetic interactions between PRP8 and PRP17/CDC40, two yeast genes involved in pre-mRNA splicing and cell cycle progression.

Genetics 154:61-71 (2000)

Sigal Ben-Yehuda , Caroline S. Russell, Ian Dix , Jean D. Beggs and Martin Kupiec

Biochemical and genetic experiments have shown that the PRP17 gene of the yeast Saccharomyces cerevisiae encodes a protein that plays a role during the second catalytic step of the splicing reaction. It was recently found that PRP17 is identical to the cell division cycle CDC40 gene. cdc40 mutants arrest at the restrictive temperature after the completion of DNA replication. Although the PRP17/CDC40 gene product is essential only at elevated temperatures, splicing intermediates accumulate in prp17 mutants even at the permissive temperature. In this report we describe extensive genetic interactions between PRP17/CDC40 and the PRP8 gene. PRP8 encodes a highly conserved U5 snRNP protein required for spliceosome assembly, and for both catalytic steps of the splicing reaction. We show that mutations in the PRP8 gene are able to suppress the temperature-sensitive growth phenotype and the splicing defect conferred by the absence of the Prp17 protein. In addition, these mutations are capable of suppressing certain alterations in the conserved PyAG trinucleotide at the 3' splice junction, as detected by an ACT1-CUP1 splicing reporter system. Moreover, other PRP8 alleles exhibit synthetic lethality with the absence of Prp17p, and show a reduced ability to splice an intron bearing an altered 3' splice junction. Based on these findings, we propose a model for the mode of interaction between the Prp8 and Prp17 proteins during the second catalytic step of the splicing reaction.


Characterization of U6 snRNA-protein interactions.

RNA 5:1470-81 (1999)

Vidal VP, Verdone L, Mayes AE, Beggs JD

Through a combination of in vitro snRNP reconstitution, photocross-linking and immunoprecipitation techniques, we have investigated the interaction of proteins with the spliceosomal U6 snRNA in U6 snRNPs, U4/U6 di-snRNPs and U4/U6.U5 tri-snRNPs. Of the seven Lsm (Sm-like) proteins that associate specifically with this spliceosomal snRNA, three were shown to contact the RNA directly, and to maintain contact as the U6 RNA is incorporated into tri-snRNPs. In tri-snRNPs, the U5 snRNP protein Prp8 contacts position 54 of U6, which is in the conserved region that contributes to the formation of the catalytic core of the spliceosome. Other tri-snRNP-specific contacts were also detected, indicating the dynamic nature of protein interactions with this important snRNA. The uridine-rich extreme 3' end of U6 RNA was shown to be essential but not sufficient for the association of the Lsm proteins. Interestingly, the Lsm proteins associate efficiently with the 3' half of U6, which contains the 3' stem-loop and uridine-rich 3' end, suggesting that the Lsm and Sm proteins may recognize similar features in RNAs. [Full text]


Characterization of Sm-like proteins in yeast and their association with U6 snRNA.

EMBO J 18:4321-31 (1999)

Mayes AE, Verdone L, Legrain P, Beggs JD

Seven Sm proteins associate with U1, U2, U4 and U5 spliceosomal snRNAs and influence snRNP biogenesis. Here we describe a novel set of Sm-like (Lsm) proteins in Saccharomyces cerevisiae that interact with each other and with U6 snRNA. Seven Lsm proteins co-immunoprecipitate with the previously characterized Lsm4p (Uss1p) and interact with each other in two-hybrid analyses. Free U6 and U4/U6 duplexed RNAs co-immunoprecipitate with seven of the Lsm proteins that are essential for the stable accumulation of U6 snRNA. Analyses of U4/U6 di-snRNPs and U4/U6.U5 tri-snRNPs in Lsm-depleted strains suggest that Lsm proteins may play a role in facilitating conformational rearrangements of the U6 snRNA in the association-dissociation cycle of spliceosome complexes. Thus, Lsm proteins form a complex that differs from the canonical Sm complex in its RNA association(s) and function. We discuss the possible existence and functions of alternative Lsm complexes, including the likelihood that they are involved in processes other than pre-mRNA splicing.