Evidence that sequence-independent binding of highly conserved U2 snRNP proteins upstream of the branch site is required for assembly of spliceosomal complex A.

Gozani, Or; Feld, R; Reed, Robin

doi:10.1101/gad.10.2.233

Cited by 229 publications

(292 citation statements)

References 27 publications

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“…Previous studies have indicated that the RES complex contacts U2 snRNP proteins 4,12 , suggesting that it might bind to the pre-mRNA in the vicinity of U2. In human spliceosomes, most U2 snRNP proteins bind to a region directly upstream of the branch site 29 , whereas in yeast only one U2 snRNP protein, Hsh155p, interacts with pre-mRNA between the branch point and the 3′ splice site 30 . To test a possibility of Snu17p interacting in this region we assembled B act∆prp2 complexes on site-specifically labeled pre-mRNAs that carried a single 32 P label directly 3′ of G nucleotides downstream of the branch site (Fig.…”

Section: Npg a R T I C L E Smentioning

confidence: 99%

Cooperative structure of the heterotrimeric pre-mRNA retention and splicing complex

Wysoczanski

Schneider

Munari

et al. 2014

Nat Struct Mol Biol

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1 1 a r t i c l e sSplicing entails the removal of introns from pre-mRNAs to generate exon-only mRNA, which is exported out of the nucleus for translation 1 . The splicing process is driven and controlled by a large and dynamic RNA-protein complex, the spliceosome 1,2 . The composition and structure of the spliceosome undergoes multiple rearrangements during a splicing reaction, in which a set of distinct structural and compositional states, designated as E, A, B act , B* and C complexes, can be defined 1 . As part of this process, small nuclear ribonucleoprotein (snRNP) particles and non-snRNP splice factors are recruited and released 1,2 . Although the organization of snRNP components of the spliceosome has received considerable attention in recent years, very little is known about the assembly, structure and dynamics of non-snRNP multimeric complexes.The non-snRNP RES complex is present in humans and yeast 3,4 . Deletion of RES genes slows splicing and leads to pre-mRNA leakage into the cytoplasm 3-5 . Distinct introns exhibit RES-dependent splicing 5-9 , as do pre-mRNAs encoding proteins functioning in RNA-nucleotide metabolism 8,10 . Components of the RES complex are found in B and C complexes of the spliceosome, in which RES can interact with U2 snRNP 4,11,12 . In yeast, the RES complex is composed of three proteins, snRNP-associated protein 17 (Snu17p, also known as Ist3p), pre-mRNA-leakage protein 1 (Pml1p) and bud siteselection protein 13 (Bud13p) 3,4 . Snu17p and Bud13p have been implicated directly in splicing 3,5,13 , whereas Pml1p has been linked to the retention of unspliced pre-mRNA in the nucleus 3,5 . Caenorhabditis elegans Bud13p is involved in embryogenesis 14 .Sequence analysis has indicated that Snu17p is a 148-residue (17.1-kDa) noncanonical member of the RRM family of proteins with a long C-terminal part, which exhibits low sequence similarity to published RRM structures 4 . Snu17p binds with nanomolar affinity to the 266-residue (30.5-kDa), natively disordered protein Bud13p 15 . The interaction has been postulated to involve a C-terminal UHM-ligand motif (ULM) in Bud13p that interacts with a U2AF-homology motif (UHM) in the RRM domain of Snu17p 13,15,16 . The only other identified domain encompasses a stretch of lysine residues at the N terminus of Bud13p. Binding of the third component, the 204-residue (23.4-kDa) Pml1p, occurs through its 50 N-terminal disordered residues. The remainder of Pml1p folds as a forkhead-associated domain 13,15,17 , which could potentially bind phosphopeptides 17 . Biochemical evidence has suggested that Snu17p acts as the central binding platform, which interacts with disordered parts of Bud13p and Pml1p 13,15,16 . The precise molecular architecture of the RES complex, however, has been elusive, and its RNA binding capabilities have remained unexplored.Here we solved the three-dimensional structure of the core of the RES complex and demonstrated that its assembly is driven by cooperativity that increases the binding affinity of the components of the complex ...

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Section: Npg a R T I C L E Smentioning

confidence: 99%

Cooperative structure of the heterotrimeric pre-mRNA retention and splicing complex

Wysoczanski

Schneider

Munari

et al. 2014

Nat Struct Mol Biol

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“…Human SF3 is involved in binding of U2 snRNP to the branch site in the splicing reaction+ The interaction between each of the SF3 subunits and the pre-mRNA sequences around the branch point sequence was analyzed by the UV-crosslink assay (Gozani et al+, 1996)+ SF3 subunits except for SAP130/SF3b130 crosslinked with the nucleotides at the 59-side of the branch site called an anchoring site+ SAP130/SF3b130 did not bind to pre-mRNA directly (Gozani et al+, 1996)+ Therefore, it is possible that SAP130/Prp12p has a role different from those of other SF3 subunits, although the exact function of each SAP in pre-mRNA splicing is still unknown+ An interesting phenotype of prp12-1 is a substratespecific splicing defect at the nonpermissive temperature+ In the analyzed pre-mRNAs, only TfIId ϩ pre-mRNA showed splicing defects at the nonpermissive temperature+ As we detected a small amount of unspliced TfIId ϩ pre-mRNA even in the wild-type cells (Fig+ 6A), TfIId ϩ pre-mRNA appears to be a substrate with low splicing efficiency in vivo+ Thus, one explanation for the substrate-specific splicing defect in prp12-1 is that mutated Prp12p produced from the prp12-1 allele retains residual activity even at the nonpermissive temperature, which does not affect splicing of efficiently spliced transcripts but does cause splicing inhibition in "poor" splicing substrates, such as TfIId ϩ FIGURE 5. A: Cytological analysis of prp12-1.…”

Section: Discussionmentioning

confidence: 99%

Mutation in the prp12 + gene encoding a homolog of SAP130/SF3b130 causes differential inhibition of pre-mRNA splicing and arrest of cell-cycle progression in Schizosaccharomyces pombe

Habara

Urushiyama

Shibuya

et al. 2001

RNA

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ABSTRACTprp12-1 is one of the mutants defective in pre-mRNA splicing at a nonpermissive temperature in Schizosaccharomyces pombe. We found that the prp12 + gene encodes a protein highly homologous with a human splicing factor, SAP130/SF3b130, a subunit of a U2 snRNP-associated complex SF3b. Prp12p was shown to interact genetically with Prp10p that is a homolog of SAP155/SF3b155, another subunit in SF3b, suggesting that Prp12p is a functional homolog of human SAP130/SF3b130. Prp12p tagged with GFP is uniformly localized in the nuclear DNA region. In addition to pre-mRNA splicing defects, the prp12-1 mutant produced elongated cells, a typical phenotype of cell division cycle (cdc) mutants, suggesting a possible link between pre-mRNA splicing and cell-cycle progression. We examined kinetics of splicing defects in prp12-1 and several other prp mutants using northern blot hybridization and found that, among all the tested pre-mRNAs, only TfIId + pre-mRNA with low splicing efficiency showed detectable splicing defects at the nonpermissive temperature in prp12-1. In addition, we found that other prp mutants with the cdc phenotype also showed differential splicing defects in tested pre-mRNAs at the nonpermissive temperature. On the other hand, prp mutants that do not exhibit the cdc phenotype showed a rapid and complete block of pre-mRNA splicing in all the tested pre-mRNAs at the nonpermissive temperature, indicating that prp mutants with weak splicing defects have a tendency to exhibit the cdc phenotype. These results suggest that the cdc phenotype in prp12-1 is caused by a selective reduction of spliced transcripts encoding a protein (or proteins) required for G2/M transition.

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“…To determine whether this protection is established prior or subsequent to exon ligation, we now report RNase H analysis of two pre-mRNAs that accumulate splicing intermediates. One RNA derived from the standard AdML splicing substrate (AdML-AG), but contains an AG → GG mutation at the 3Ј splice site (AdML-GG; Gozani et al 1996;Anderson and Moore 1997). The other (␣-TM) derived from E2-I2-E3 of rat ␣-tropomyosin (Smith et al 1989) and was truncated upstream of the 3Ј splice site.…”

Section: Extensive Rnase Protection Of the 5ј Exon Intermediatementioning

confidence: 99%

5′ exon interactions within the human spliceosome establish a framework for exon junction complex structure and assembly

Reichert

Hir²,

Jurica³

et al. 2002

Genes Dev.

126

137

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A general consequence of pre-mRNA splicing is the stable deposition of several proteins 20-24 nucleotides (nt) upstream of exon-exon junctions on spliced mRNAs. This exon junction complex (EJC) contains factors involved in mRNA export, cytoplasmic localization, and nonsense-mediated mRNA decay. Here we probed the mechanism and timing of EJC assembly. Over the course of splicing, the 5 exon is subject to numerous dynamic protein-RNA interactions involving at least nine distinct polypeptides. Within the fully assembled spliceosome, these interactions afford protection to the last 25-27 nt of the 5 exon intermediate. Throughout their lifetimes in vivo, mRNAs exist as mRNA-protein particles (mRNPs). The associated proteins control every aspect of mRNA metabolism, from subcellular transport to translational efficiency to rate of decay. Exactly which proteins associate with a particular mRNA depends on its sequence, its subcellular localization, and its synthetic history. Furthermore, the complement of mRNP proteins evolves as the mRNA moves to different locations and is acted on by such processes as nuclear export and translation. Many proteins join the mRNP in the nucleus and then accompany it to the cytoplasm, where they influence subsequent mRNA metabolism ( These splicing-specific mRNP proteins constitute the exon junction complex (EJC), which associates with spliced mRNAs in a sequence-independent manner a fixed distance upstream of exon-exon junctions (Le Hir et al. 2000b). Using coimmunoprecipitation strategies, we and others recently reported that the EJC assembled in HeLa nuclear extract contains numerous epitopes, including those recognized by antibodies against REF/Aly, Y14, SRm160, DEK, RNPS1, UAP56, and Magoh (Blencowe et al. 1998;Mayeda et al. 1999;Kataoka et al. 2000Kataoka et al. , 2001Le Hir et al. 2000a,b, 2001bMcGarvey et al. 2000;Zhou et al. 2000;Gatfield et al. 2001;Luo et al. 2001). When assembled in vivo, the EJC is additionally precipitated by antibodies against Upf2, Upf3, and the TAP:p15 heterodimer (Kim et al. 2001b;Le Hir et al. 2001a;Lykke-Andersen et al. 2001). Finally, REF/Aly, Y14, SRm160, RNPS1, Upf2, Upf3, and TAP were all present in mRNPs immunopurified from the nuclear fraction of COS cells with antibodies against a subunit of the nuclear cap binding complex (Lejeune et al. 2002).The known EJC components function at multiple levels of mRNA metabolism. SRm160 and RNPS1 were originally characterized as activators of pre-mRNA splicing (Blencowe et al. 1998;Mayeda et al. 1999

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Evidence that sequence-independent binding of highly conserved U2 snRNP proteins upstream of the branch site is required for assembly of spliceosomal complex A.

Cited by 229 publications

References 27 publications

Cooperative structure of the heterotrimeric pre-mRNA retention and splicing complex

Cooperative structure of the heterotrimeric pre-mRNA retention and splicing complex

Mutation in the prp12 + gene encoding a homolog of SAP130/SF3b130 causes differential inhibition of pre-mRNA splicing and arrest of cell-cycle progression in Schizosaccharomyces pombe

5′ exon interactions within the human spliceosome establish a framework for exon junction complex structure and assembly

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