Crystal Structure of the Pml1p Subunit of the Yeast Precursor mRNA Retention and Splicing Complex

The precursor mRNA retention and splicing (RES) complex mediates nuclear retention and enhances splicing of precursor mRNAs. The RES complex from yeast comprises three proteins, Snu17p, Bud13p and Pml1p. Snu17p acts as a central platform that concomitantly binds the Bud13p and Pml1p subunits via short peptide epitopes. As a step to decipher the molecular architecture of the RES complex, we have determined crystal structures of full-length Pml1p and N-terminally truncated Pml1p. The first 50 residues of full-length Pml1p, encompassing the Snu17p-binding region, are disordered, showing that Pml1p binds to Snu17p via an intrinsically unstructured region. The remainder of Pml1p folds as a forkhead-associated (FHA) domain, which is expanded by a number of noncanonical elements compared with known FHA domains from other proteins. An atypical N-terminal appendix runs across one β-sheet and thereby stabilizes the domain as shown by deletion experiments. FHA domains are thought to constitute phosphopeptide-binding elements. Consistently, a sulfate ion was found at the putative phosphopeptide-binding loops of full-length Pml1p. The N-terminally truncated version of the protein lacked a similar phosphopeptide mimic but retained an almost identical structure. A long loop neighboring the putative phosphopeptide-binding site was disordered in both structures. Comparison with other FHA domain proteins suggests that this loop adopts a defined conformation upon ligand binding and thereby confers ligand specificity. Our results show that in the RES complex, an FHA domain of Pml1p is flexibly tethered via an unstructured N-terminal region to Snu17p.     

An unusual RNA recognition motif acts as a scaffold for multiple proteins in the pre-mRNA retention and splicing complex

The yeast pre-mRNA retention and splicing complex counteracts the escape of unspliced pre-mRNAs from the nucleus and activates splicing of a subset of Mer1p-dependent genes. A homologous complex is present in activated human spliceosomes. In many components of the spliceosome, RNA recognition motifs (RRMs) serve as versatile protein-RNA or protein-protein interaction platforms. Here, we show that in the retention and splicing complex, an atypical RRM of the Snu17p (small nuclear ribonucleoprotein-associated protein 17) subunit acts as a scaffold that organizes the other two constituents, Bud13p (bud site selection 13) and Pml1p (pre-mRNA leakage 1). GST pull-down experiments and size exclusion chromatography revealed that Snu17p constitutes the central platform of the complex, whereas Bud13p and Pml1p do not interact with each other. Fluorimetric structure probing showed the entire Bud13p and the N-terminal third of Pml1p to be natively disordered in isolation. Mutational analysis and tryptophan fluorescence confirmed that a conserved tryptophan-containing motif in the C terminus of Bud13p binds to the core RRM of Snu17p, whereas a different interaction surface encompassing a C-terminal extension of the Snu17p RRM is required to bind an N-terminal peptide of Pml1p. Isothermal titration calorimetry revealed 1:1 interaction stoichiometries, large negative binding entropies, and dissociation constants in the low nanomolar and micromolar ranges for the Snu17p-Bud13p and the Snu17p-Pml1p interactions, respectively. Our results demonstrate that the noncanonical Snu17p RRM concomitantly binds multiple ligand proteins via short, intrinsically unstructured peptide epitopes and thereby acts as a platform that displays functional modules of the ligands, such as a forkhead-associated domain of Pml1p and a conserved polylysine motif of Bud13p.     

Structure of the yeast Pml1 splicing factor and its integration into the RES complex

The RES complex was previously identified in yeast as a splicing factor affecting nuclear pre-mRNA retention. This complex was shown to contain three subunits, namely Snu17, Bud13 and Pml1, but its mode of action remains ill-defined. To obtain insights into its function, we have performed a structural investigation of this factor. Production of a short N-terminal truncation of residues that are apparently disordered allowed us to determine the X-ray crystallographic structure of Pml1. This demonstrated that it consists mainly of a FHA domain, a fold which has been shown to mediate interactions with phosphothreonine-containing peptides. Using a new sensitive assay based on alternative splice-site choice, we show, however, that mutation of the putative phosphothreonine-binding pocket of Pml1 does not affect pre-mRNA splicing. We have also investigated how Pml1 integrates into the RES complex. Production of recombinant complexes, combined with serial truncation and mutagenesis of their subunits, indicated that Pml1 binds to Snu17, which itself contacts Bud13. This analysis allowed us to demarcate the binding sites involved in the formation of this assembly. We propose a model of the organization of the RES complex based on these results, and discuss the functional consequences of this architecture.     

Proteomic analysis identifies a new complex required for nuclear pre-mRNA retention and splicing

Using the proteomic tandem affinity purification (TAP) method, we have purified the Saccharomyces cerevisie U2 snRNP-associated splicing factors SF3a and SF3b. While SF3a purification revealed only the expected subunits Prp9p, Prp11p and Prp21p, yeast SF3b was found to contain only six subunits, including previously known components (Rse1p, Hsh155p, Cus1p, Hsh49p), the recently identified Rds3p factor and a new small essential protein (Ysf3p) encoded by an unpredicted split ORF in the yeast genome. Surprisingly, Snu17p, the proposed yeast orthologue of the seventh human SF3b subunit, p14, was not found in the yeast complex. TAP purification revealed that Snu17p, together with Bud13p and a newly identified factor, Pml1p/Ylr016c, form a novel trimeric complex. Subunits of this complex were not essential for viability. However, they are required for efficient splicing in vitro and in vivo. Furthermore, inactivation of this complex causes pre-mRNA leakage from the nucleus. The corresponding complex was named pre-mRNA REtention and Splicing (RES). The presence of RES subunit homologues in numerous eukaryotes suggests that its function is evolutionarily conserved.     

Dynamic Contacts of U2, RES,Cwc25, Prp8 and Prp45 Proteins with the Pre-mRNA Branch-Site and 3' Splice Site during Catalytic Activation and Step 1 Catalysis in Yeast Spliceosomes

Little is known about contacts in the spliceosome between proteins and intron nucleotides surrounding the pre-mRNA branch-site and their dynamics during splicing. We investigated protein-pre-mRNA interactions by UV-induced crosslinking of purified yeast B^act spliceosomes formed on site-specifically labeled pre-mRNA, and analyzed their changes after conversion to catalytically-activated B* and step 1 C complexes, using a purified splicing system. Contacts between nucleotides upstream and downstream of the branch-site and the U2 SF3a/b proteins Prp9, Prp11, Hsh49, Cus1 and Hsh155 were detected, demonstrating that these interactions are evolutionarily conserved. The RES proteins Pml1 and Bud13 were shown to contact the intron downstream of the branch-site. A comparison of the B^act crosslinking pattern versus that of B* and C complexes revealed that U2 and RES protein interactions with the intron are dynamic. Upon step 1 catalysis, Cwc25 contacts with the branch-site region, and enhanced crosslinks of Prp8 and Prp45 with nucleotides surrounding the branch-site were observed. Cwc25’s step 1 promoting activity was not dependent on its interaction with pre-mRNA, indicating it acts via protein-protein interactions. These studies provide important insights into the spliceosome''s protein-pre-mRNA network and reveal novel RNP remodeling events during the catalytic activation of the spliceosome and step 1 of splicing.     

Interactions of the yeast SF3b splicing factor

The U2 snRNP promotes prespliceosome assembly through interactions that minimally involve the branchpoint binding protein, Mud2p, and the pre-mRNA. We previously showed that seven proteins copurify with the yeast (Saccharomyces cerevisiae) SF3b U2 subcomplex that associates with the pre-mRNA branchpoint region: Rse1p, Hsh155p, Hsh49p, Cus1p, and Rds3p and unidentified subunits p10 and p17. Here proteomic and genetic studies identify Rcp10p as p10 and show that it contributes to SF3b stability and is necessary for normal cellular Cus1p accumulation and for U2 snRNP recruitment in splicing. Remarkably, only the final 53 amino acids of Rcp10p are essential. p17 is shown to be composed of two accessory splicing factors, Bud31p and Ist3p, the latter of which independently associates with the RES complex implicated in the nuclear pre-mRNA retention. A directed two-hybrid screen reveals a network of prospective interactions that includes previously unreported intra-SF3b contacts and SF3b interactions with the RES subunit Bud13p, the Prp5p DExD/H-box protein, Mud2p, and the late-acting nineteen complex. These data establish the concordance of yeast and mammalian SF3b complexes, implicate accessory splicing factors in U2 snRNP function, and support SF3b contribution from early pre-mRNP recognition to late steps in splicing.     

Assignment of 1H, 13C, and 15N resonances of the RNA binding protein Snu13p from Saccharomyces cerevisiae

Snu13p is a highly conserved RNA binding protein from Saccharomyces cerevisiae required for both eukaryotic pre-mRNA splicing and pre-rRNA processing. The ¹H, ¹³C, and ¹⁵N assignments were determined from multidimensional, multinuclear NMR experiments conducted at 25°C.     

The ribosomal translocase homologue Snu114p is involved in unwinding U4/U6 RNA during activation of the spliceosome

Snu114p is a yeast U5 snRNP protein homologous to the ribosomal elongation factor EF-2. Snu114p exhibits the same domain structure as EF-2, including the G-domain, but with an additional N-terminal domain. To test whether Snu114p in the spliceosome is involved in rearranging RNA secondary structures (by analogy to EF-2 in the ribosome), we created conditionally lethal mutants. Deletion of this N-terminal domain (snu114ΔN) leads to a temperature-sensitive phenotype at 37°C and a pre-mRNA splicing defect in vivo. Heat treatment of snu114ΔN extracts blocked splicing in vitro before the first step. The snu114ΔN still associates with the tri-snRNP, and the stability of this particle is not significantly impaired by thermal inactivation. Heat treatment of snu114ΔN extracts resulted in accumulation of arrested spliceosomes in which the U4 RNA was not efficiently released, and we show that U4 is still base paired with the U6 RNA. This suggests that Snu114p is involved, directly or indirectly, in the U4/U6 unwinding, an essential step towards spliceosome activation.     

Identification by mass spectrometry and functional analysis of novel proteins of the yeast [U4/U6.U5] tri-snRNP.

The 25S [U4/U6.U5] tri-snRNP (small nuclear ribonucleoprotein) is a central unit of the nuclear pre-mRNA splicing machinery. The U4, U5 and U6 snRNAs undergo numerous rearrangements in the spliceosome, and knowledge of all of the tri-snRNP proteins is crucial to the detailed investigation of the RNA dynamics during the spliceosomal cycle. Here we characterize by mass spectrometric methods the proteins of the purified [U4/U6.U5] tri-snRNP from the yeast Saccharomyces cerevisiae. In addition to the known tri-snRNP proteins (only one, Lsm3p, eluded detection), we identified eight previously uncharacterized proteins. These include four Sm-like proteins (Lsm2p, Lsm5p, Lsm6p and Lsm7p) and four specific proteins named Snu13p, Dib1p, Snu23p and Snu66p. Snu13p comprises a putative RNA-binding domain. Interestingly, the Schizosaccharomyces pombe orthologue of Dib1p, Dim1p, was previously assigned a role in cell cycle progression. The role of Snu23p, Snu66p and, additionally, Spp381p in pre-mRNA splicing was investigated in vitro and/or in vivo. Finally, we show that both tri-snRNPs and the U2 snRNP are co-precipitated with protein A-tagged versions of Snu23p, Snu66p and Spp381p from extracts fractionated by glycerol gradient centrifugation. This suggests that these proteins, at least in part, are also present in a [U2.U4/U6.U5] tetra-snRNP complex.     

Conformational Preferences of Modified Nucleoside N(4)-Acetylcytidine, ac4C Occur at “Wobble” 34th Position in the Anticodon Loop of tRNA

Conformational preferences of modified nucleoside, N(4)-acetylcytidine, ac⁴C have been investigated using quantum chemical semi-empirical RM1 method. Automated geometry optimization using PM3 method along with ab initio methods HF SCF (6-31G**), and density functional theory (DFT; B3LYP/6-31G**) have also been made to compare the salient features. The most stable conformation of N(4)-acetyl group of ac⁴C prefers “proximal” orientation. This conformation is stabilized by intramolecular hydrogen bonding between O(7)···HC(5), O(2)···HC2′, and O4′···HC(6). The “proximal” conformation of N(4)-acetyl group has also been observed in another conformational study of anticodon loop of E. coli elongator tRNA^Met. The solvent accessible surface area (SASA) calculations revealed the role of ac⁴C in anticodon loop. The explicit molecular dynamics simulation study also shows the “proximal” orientation of N(4)-acetyl group. The predicted “proximal” conformation would allow ac⁴C to interact with third base of codon AUG/AUA whereas the ‘distal’ orientation of N(4)-acetyl cytidine side-chain prevents such interactions. Single point energy calculation studies of various models of anticodon–codon bases revealed that the models ac⁴C₍₃₄₎(Proximal):G₃, and ac⁴C₍₃₄₎(Proximal):A₃ are energetically more stable as compared to models ac⁴C₍₃₄₎(Distal):G₃, and ac⁴C₍₃₄₎(Distal):A₃, respectively. MEPs calculations showed the unique potential tunnels between the hydrogen bond donor–acceptor atoms of ac⁴C₍₃₄₎(Proximal):G₃/A₃ base pairs suggesting role of ac⁴C in recognition of third letter of codons AUG/AUA. The “distal” conformation of ac⁴C might prevent misreading of AUA codon. Hence, this study could be useful to understand the role of ac⁴C in the tertiary structure folding of tRNA as well as in the proper recognition of codons during protein biosynthesis process.     

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

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.     

An evolutionarily conserved U5 snRNP-specific protein is a GTP-binding factor closely related to the ribosomal translocase EF-2.

The driving forces behind the many RNA conformational changes occurring in the spliceosome are not well understood. Here we characterize an evolutionarily conserved human U5 small nuclear ribonucleoprotein (snRNP) protein (U5-116kD) that is strikingly homologous to the ribosomal elongation factor EF-2 (ribosomal translocase). A 114 kDa protein (Snu114p) homologous to U5-116kD was identified in Saccharomyces cerevisiae and was shown to be essential for yeast cell viability. Genetic depletion of Snu114p results in accumulation of unspliced pre-mRNA, indicating that Snu114p is essential for splicing in vivo. Antibodies specific for U5-116kD inhibit pre-mRNA splicing in a HeLa nuclear extract in vitro. In HeLa cells, U5-116kD is located in the nucleus and colocalizes with snRNP-containing subnuclear structures referred to as speckles. The G domain of U5-116kD/Snu114p contains the consensus sequence elements G1-G5 important for binding and hydrolyzing GTP. Consistent with this, U5-116kD can be cross-linked specifically to GTP by UV irradiation of U5 snRNPs. Moreover, a single amino acid substitution in the G1 sequence motif of Snu114p, expected to abolish GTP-binding activity, is lethal, suggesting that GTP binding and probably GTP hydrolysis is important for the function of U5-116kD/Snu114p. This is to date the first evidence that a G domain-containing protein plays an essential role in the pre-mRNA splicing process.     

A Highlights from MBoC Selection: Dual Functions of Yeast tRNA Ligase in the Unfolded Protein Response: Unconventional Cytoplasmic Splicing of HAC1 Pre-mRNA Is Not Sufficient to Release Translational Attenuation

The unfolded protein response (UPR) is an essential signal transduction to cope with protein-folding stress in the endoplasmic reticulum. In the yeast UPR, the unconventional splicing of HAC1 mRNA is a key step. Translation of HAC1 pre-mRNA (HAC1^u mRNA) is attenuated on polysomes and restarted only after splicing upon the UPR. However, the precise mechanism of this restart remained unclear. Here we show that yeast tRNA ligase (Rlg1p/Trl1p) acting on HAC1 ligation has an unexpected role in HAC1 translation. An RLG1 homologue from Arabidopsis thaliana (AtRLG1) substitutes for yeast RLG1 in tRNA splicing but not in the UPR. Surprisingly, AtRlg1p ligates HAC1 exons, but the spliced mRNA (HAC1ⁱ mRNA) is not translated efficiently. In the AtRLG1 cells, the HAC1 intron is circularized after splicing and remains associated on polysomes, impairing relief of the translational repression of HAC1ⁱ mRNA. Furthermore, the HAC1 5′ UTR itself enables yeast Rlg1p to regulate translation of the following ORF. RNA IP revealed that yeast Rlg1p is integrated in HAC1 mRNP, before Ire1p cleaves HAC1^u mRNA. These results indicate that the splicing and the release of translational attenuation of HAC1 mRNA are separable steps and that Rlg1p has pivotal roles in both of these steps.     

Analysis of Snu13p mutations reveals differential interactions with the U4 snRNA and U3 snoRNA

Pre-mRNA splicing is executed by the spliceosome, a complex of small nuclear RNAs (snRNAs) and numerous proteins. One such protein, 15.5K/Snu13p, is associated with the spliceosomal U4/U6.U5 tri-snRNP and box C/D small nucleolar ribonucleoprotein particles (snoRNPs), which act during preribosomal RNA (rRNA) processing. As such, it is the first splicing factor to be identified in two functionally distinct particles. 15.5K binds to an internal helix-bulge-helix (K-turn) structure in the U4 snRNA and two such structures in the U3 snoRNA. Previous work has concentrated on the structural basis of the interaction of 15.5K with the RNAs and has been carried out in vitro. Here we present a functional analysis of Snu13p in vivo, using a galactose inducible SNU13 strain to investigate the basis of three lethal mutations in Saccharomyces cerevisiae. Two are point mutations that map to the RNA-binding domain, and the third is a C-terminal deletion. These mutations result in accumulation of unspliced pre-mRNA, confirming a role for Snu13p in pre-mRNA splicing. In addition, these mutants also display rRNA processing defects that are variable in nature. Analysis of one mutant in the RNA-binding domain reveals a reduction in the levels of the U4 snRNA, U6 snRNA, and box C/D snoRNAs, but not H/ACA snoRNAs, supporting a role for Snu13p in accumulation and/or maintenance of specific RNAs. The mutations in the RNA-binding domain exhibit differential binding to the U4 snRNA and U3 snoRNA in vitro, suggesting that there are differences in the mode of interaction of Snu13p with these two RNAs.     

A novel yeast U2 snRNP protein, Snu17p, is required for the first catalytic step of splicing and for progression of spliceosome assembly

We have isolated and microsequenced Snu17p, a novel yeast protein with a predicted molecular mass of 17 kDa that contains an RNA recognition motif. We demonstrate that Snu17p binds specifically to the U2 small nuclear ribonucleoprotein (snRNP) and that it is part of the spliceosome, since the pre-mRNA and the lariat-exon 2 are specifically coprecipitated with Snu17p. Although the SNU17 gene is not essential, its knockout leads to a slow-growth phenotype and to a pre-mRNA splicing defect in vivo. In addition, the first step of splicing is dramatically decreased in extracts prepared from the snu17 deletion (snu17Delta) mutant. This defect is efficiently reversed by the addition of recombinant Snu17p. To investigate the step of spliceosome assembly at which Snu17p acts, we have used nondenaturing gel electrophoresis. In Snu17p-deficient extracts, the spliceosome runs as a single slowly migrating complex. In wild-type extracts, usually at least two distinct complexes are observed: the prespliceosome, or B complex, containing the U2 but not the U1 snRNP, and the catalytically active spliceosome, or A complex, containing the U2, U6, and U5 snRNPs. Northern blot analysis and affinity purification of the snu17Delta spliceosome showed that it contains the U1, U2, U6, U5, and U4 snRNPs. The unexpected stabilization of the U1 snRNP and the lack of dissociation of the U4 snRNP suggest that loss of Snu17p inhibits the progression of spliceosome assembly prior to U1 snRNP release and after [U4/U6.U5] tri-snRNP addition.     

The Saccharomyces cerevisiae TAN1 gene is required for N4-acetylcytidine formation in tRNA

The biogenesis of transfer RNA is a process that requires many different factors. In this study, we describe a genetic screen aimed to identify gene products participating in this process. By screening for mutations lethal in combination with a sup61-T47:2C allele, coding for a mutant form of, the nonessential TAN1 gene was identified. We show that the TAN1 gene product is required for formation of the modified nucleoside N(4)-acetylcytidine (ac(4)C) in tRNA. In Saccharomyces cerevisiae, ac(4)C is present at position 12 in tRNAs specific for leucine and serine as well as in 18S ribosomal RNA. Analysis of RNA isolated from a tan1-null mutant revealed that ac(4)C was absent in tRNA, but not rRNA. Although no tRNA acetyltransferase activity by a GST-Tan1 fusion protein was detected, a gel-shift assay revealed that Tan1p binds tRNA, suggesting a direct role in synthesis of ac(4)C(12). The absence of the TAN1 gene in the sup61-T47:2C mutant caused a decreased level of mature, indicating that ac(4)C(12) and/or Tan1p is important for tRNA stability.     

prpf4 is essential for cell survival and posterior lateral line primordium migration in zebrafish

Prpf4 (pre-mRNA processing factor 4), a key component of spliceosome, plays critical roles in pre-mRNA splicing and its mutations result in retinitis pigmentosa due to photoreceptor defects. In this study, we characterized a zebrafish prpf4^t243 mutant harboring a Tol2 transposon-based gene trap cassette in the third intron of the prpf4 gene. Cells in the brain and spinal cord gradually undergo p53-dependent apoptosis after 28 hpf in prpf4^t243 mutants, suggesting that a widespread function of prpf4 in neural cell survival. In addition, prpf4 is essential for survival of posterior lateral line primordial (pLLP) cells. prpf4 deficiency perturbs Fgf, Wnt/β-catenin and chemokine signaling pathways and impairs pLLP migration. RNA-Seq analysis suggests that prpf4 deficiency may impair spliceosome assembly, leading to compensatory upregulation of core spliceosomal genes and alteration of pre-mRNA splicing. Taken together, our studies uncover an essential role of prpf4 in pre-mRNA splicing, cell survival and pLLP migration.