Structural Basis for the Molecular Recognition between Human Splicing Factors U2AF65 and SF1/mBBP

Selenko, Philipp; Gregorovic, Goran; Sprangers, Remco; Stier, Günter; Rhani, Zakaria; Krämer, Angela; Sattler, Michael

doi:10.1016/s1097-2765(03)00115-1

Cited by 195 publications

(312 citation statements)

References 49 publications

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“…Furthermore, the analogous substitution in the spU2AF SM ⌿RRM was also lethal, and our data indicate that this domain does contribute to RNA binding (Webb and Wise, 2004). The discrepancy can be reconciled by structural data implicating the hsU2AF LG amino acid analogous to F476 in packing helix C against the ␤-sheet that contains the RNP1-like sequence (Selenko et al, 2003), which would block the potential RNA binding surface, whereas the small subunit of U2AF does not have a recognizable helix C (Kielkopf et al, 2001). The inability of the [F476D, Y479D] substitution to support growth also echoes the effect of the corresponding mutation in the ⌿RRM of the S. cerevisiae large subunit orthologue MUD2.…”

Section: U2af Function In Vivomentioning

confidence: 80%

“…First, because hsU2AF LG binds to and collaborates with SF1/BBP (Berglund et al, 1998;Selenko et al, 2003), we first asked whether the strength of the branchpoint correlates, either directly or inversely, with spU2AF-dependence; no relation was found between the match to the branchpoint consensus (Zhang and Marr, 1994) and spU2AF requirements (see Figure 6). Second, given that introns are bridged by a complex anchored at one end by the U1 snRNP and at the other by U2AF (Abovich et al, 1994;Berglund et al, 1998), we examined the 5Ј splice sites of the 22 introns; there was also no correlation between the strength of the 5Ј splice site and spU2AF requirements (see Figure 6).…”

Section: U2af-dependence Does Not Correlate With Other Obvious Featurmentioning

confidence: 99%

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Analysis of Mutant Phenotypes and Splicing Defects Demonstrates Functional Collaboration between the Large and Small Subunits of the Essential Splicing Factor U2AF In Vivo

Webb

Lakhe-Reddy

Romfo

et al. 2005

MBoC

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The heterodimeric splicing factor U2AF plays an important role in 3 splice site selection, but the division of labor between the two subunits in vivo remains unclear. In vitro assays led to the proposal that the human large subunit recognizes 3 splice sites with extensive polypyrimidine tracts independently of the small subunit. We report in vivo analysis demonstrating that all five domains of spU2AF LG are essential for viability; a partial deletion of the linker region, which forms the small subunit interface, produces a severe growth defect and an aberrant morphology. A small subunit zinc-binding domain mutant confers a similar phenotype, suggesting that the heterodimer functions as a unit during splicing in Schizosaccharomyces pombe. As this is not predicted by the model for metazoan 3 splice site recognition, we sought introns for which the spU2AF LG and spU2AF SM make distinct contributions by analyzing diverse splicing events in strains harboring mutations in each partner. Requirements for the two subunits are generally parallel and, moreover, do not correlate with the length or strength of the 3 pyrimidine tract. These and other studies performed in fission yeast support a model for 3 splice site recognition in which the two subunits of U2AF functionally collaborate in vivo. INTRODUCTIONThe removal of noncoding introns and the joining of coding exons via splicing, an essential step in the eukaryotic premRNA processing pathway, requires accurate splice site selection by the spliceosome. Initial recognition of the 5Ј exon/intron boundary is achieved via base pairing with the U1 snRNA component of the U1 snRNP (Zhuang and Weiner, 1986), whereas U2AF (U2 snRNP auxiliary factor) is the first factor bound to the 3Ј splice site (Ruskin et al., 1988). Biochemical complementation assays demonstrated that U2AF is required for the subsequent ATP-dependent association of U2 snRNP with pre-mRNA branchpoints (Ruskin et al., 1988;Valcarcel et al., 1996). Purified U2AF is a heterodimer composed of large and small subunits in humans (Zamore and Green, 1989), Drosophila melanogaster (Kanaar et al., 1993;Rudner et al., 1996), Caenorhabditis elegans (Zorio et al., 1997; Zorio and Blumenthal, 1999) and Schizosaccharomyces pombe (Potashkin et al., 1993;Wentz-Hunter and Potashkin, 1996). Functional conservation of this splicing factor is evidenced by 1) the restoration of splicing activity to U2AF-depleted HeLa nuclear splicing extracts via addition of Drosophila U2AF large subunit (dmU2AF LG ; Zamore and Green, 1991) and 2) the ability of human U2AF 35 (hsU2AF SM ) to restore growth to an S. pombe strain lacking the small subunit (Webb and Wise, 2004).The structural domains of U2AF are also conserved except in Saccharomyces cerevisiae, where the large subunit is highly divergent and the small subunit is absent entirely (Abovich et al., 1994). The small subunit of U2AF consists of two zinc-binding domains (ZBDs) surrounding a central pseudo-RNA recognition motif (⌿RRM; Rudner et al., 1998b), also known as a PUMP (PUF60/U2AF/M...

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Section: U2af Function In Vivomentioning

confidence: 80%

Section: U2af-dependence Does Not Correlate With Other Obvious Featurmentioning

confidence: 99%

Analysis of Mutant Phenotypes and Splicing Defects Demonstrates Functional Collaboration between the Large and Small Subunits of the Essential Splicing Factor U2AF In Vivo

Webb

Lakhe-Reddy

Romfo

et al. 2005

MBoC

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“…Sequence homology of Bud13p to ligands that bind to U2AF-homology motifs of RRM domains suggested that Bud13p interacts with Snu17p according to canonical UHM-ULM interactions (Fig. 6b,d) 13,15,16,[18][19][20][21][22] . However, the three-dimensional structure of the RES core complex demonstrated a distinct interaction mode unlike that of UHM and ULM (Fig.…”

Section: Discussionmentioning

confidence: 99%

“…1a and 2b). In canonical UHM-ULM interactions, the ULM is formed by a positively charged sequence followed by an essential tryptophan [18][19][20][21][22] . In Bud13p the tryptophan is located at position 232, and it was supposed to bind to the hydrophobic pocket between α-helices 1 and 2 (refs.…”

Section: Structure Of the Res Core Complexmentioning

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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“…The N-terminal region of the 68 kD subunit contains an RNP-type RNA binding domain (RBD), which is necessary for binding to the 25 kD subunit [164]. RBDs have been observed in protein-protein interactions in other cases, for example the splicing factor U2AF [165,166] and the Drosophila proteins Y14 and Mago [167]. The C-terminal region of the 68 kD subunit is rich in RS, RD and RE repeats that are similar to pre-mRNA splicing SR proteins.…”

Section: Mammalian Cleavage Factor I (Cf I M )mentioning

confidence: 99%

Protein factors in pre-mRNA 3′-end processing

Mandel

Bai

Tong

2007

Cell. Mol. Life Sci.

351

466

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Most eukaryotic mRNA precursors (pre-mRNAs) must undergo extensive processing, including cleavage and polyadenylation at the 3′-end. Processing at the 3′-end is controlled by sequence elements in the pre-mRNA (cis elements) as well as protein factors. Despite the seeming biochemical simplicity of the processing reactions, more than 14 proteins have been identified for the mammalian complex, and more than 20 proteins have been identified for the yeast complex. The 3′-end processing machinery also has important roles in transcription and splicing. The mammalian machinery contains several sub-complexes, including cleavage and polyadenylation specificity factor (CPSF), cleavage stimulation factor (CstF), cleavage factor I (CF I m ), and cleavage factor II (CF II m ). Additional protein factors include poly(A) polymerase (PAP), poly(A) binding protein (PABP), symplekin, and the C-terminal domain (CTD) of RNA polymerase II largest subunit. The yeast machinery includes cleavage factor IA (CF IA), cleavage factor IB (CF IB), and cleavage and polyadenylation factor (CPF).

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Structural Basis for the Molecular Recognition between Human Splicing Factors U2AF65 and SF1/mBBP

Cited by 195 publications

References 49 publications

Analysis of Mutant Phenotypes and Splicing Defects Demonstrates Functional Collaboration between the Large and Small Subunits of the Essential Splicing Factor U2AF In Vivo

Analysis of Mutant Phenotypes and Splicing Defects Demonstrates Functional Collaboration between the Large and Small Subunits of the Essential Splicing Factor U2AF In Vivo

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

Protein factors in pre-mRNA 3′-end processing

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