Solution Structure of the N-terminal RNP Domain of U1A Protein: The Role of C-terminal Residues in Structure Stability and RNA Binding

Avis, Johanna M.; Allain, Frédéric H.‐T.; Howe, Peter W.A.; Varani, Gabriele; Nagai, Kiyoshi; Neuhaus, David

doi:10.1006/jmbi.1996.0171

Cited by 167 publications

(224 citation statements)

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“…4A). In one orientation, the ␤-sheet of the RRM is masked (19); an Ϸ135°rotation of this helix reveals the surface of the RRM (20) in a structure similar to that observed in the U1A protein⅐RNA complex (12). In contrast to U1A, the C-terminal helices of p14 are held rigidly in position through an extensive network of hydrophobic and hydrophilic interactions, which are both intramolecular and between p14 and SF3b (Figs.…”

Section: Rna Protein Interactions In a Minimalmentioning

confidence: 76%

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Crystal structure of a core spliceosomal protein interface

Schellenberg

Edwards

Ritchie

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

112

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The precise excision of introns from precursor mRNAs (pre-mRNAs) in eukaryotes is accomplished by the spliceosome, a complex assembly containing five small nuclear ribonucleoprotein (snRNP) particles. Human p14, a component of the spliceosomal U2 and U11͞U12 snRNPs, has been shown to associate directly with the pre-mRNA branch adenosine early in spliceosome assembly and within the fully assembled spliceosome. Here we report the 2.5-Å crystal structure of a complex containing p14 and a peptide derived from the p14-associated U2 snRNP component SF3b155. p14 contains an RNA recognition motif (RRM), the surface of which is largely occluded by a C-terminal ␣-helix and a portion of the SF3b155 peptide. An analysis of RNA⅐protein crosslinking to wildtype and mutant p14 shows that the branch adenosine directly interacts with a conserved aromatic within a pocket on the surface of the complex. This result, combined with a comparison of the structure with known RRMs and pseudoRRMs as well as modelbuilding by using the electron cryomicroscopy structure of a spliceosomal U11͞U12 di-snRNP, suggests that p14⅐SF3b155 presents a noncanonical surface for RNA recognition at the heart of the mammalian spliceosome.RNA-binding proteins ͉ RNA splicing ͉ spliceosome P recursor mRNA (pre-mRNA) splicing occurs through two sequential transesterification reactions. The first step involves displacement of the 5Ј exon by the nucleophilic attack of the 2Ј hydroxyl of the conserved branch region adenosine. In the second step, the free 5Ј exon attacks the 3Ј splice site to yield the ligated mRNA product. Assembly of the spliceosomal catalytic machinery containing the U1, U2, and U4͞U5͞U6 small nuclear ribonucleoproteins (snRNPs) is directed by conserved sequence elements at the splice sites and within the intron including the branch region (1-3). Spliceosome assembly occurs in an ordered, stepwise fashion through a series of discrete intermediates and involves sequential steps of pre-mRNA recognition by protein and snRNA components of the spliceosome as well as formation and rearrangement of snRNA⅐snRNA interactions. The fully assembled spliceosome contains a U2͞U6 snRNA structure that has been proposed to form the active site for catalysis of the transesterifications (4, 5). Although pre-mRNA splicing is therefore believed to be intrinsically RNA catalyzed, RNA⅐protein crosslinking studies have established direct association of human p14 with the reactive branch adenosine of the pre-mRNA (6, 7). Thus, the catalytic heart of the spliceosome may include protein as well as RNA components.Initial recognition of the pre-mRNA includes a stable U1 snRNP⅐5Ј splice-site base pairing and the association of nonsnRNP protein factors with the branch region͞3Ј splice site to form the early or commitment complex. The ATP-dependent transition to A complex involves the stable association of U2 snRNP with the pre-mRNA, an interaction that includes the formation of a duplex between U2 snRNA and the pre-mRNA branch region. The bulging of the branch adenosine from...

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Section: Rna Protein Interactions In a Minimalmentioning

confidence: 76%

“…The structure of the p14⅐peptide complex is reminiscent of a number of other unusual RRM and RRM-like structures (19)(20)(21)(22). An ␣-helix C-terminal to the RRM of the spliceosomal U1A protein has been shown to adopt two different conformations with respect to the RNAbinding face of the RRM (Fig.…”

Section: Rna Protein Interactions In a Minimalmentioning

confidence: 99%

Crystal structure of a core spliceosomal protein interface

Schellenberg

Edwards

Ritchie

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

112

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“…Deletion of this region abolished binding of Pml1p to Snu17p 13,15 . RRM extensions, as observed in Snu17p, are already folded in an RRM's free state [34][35][36] or fold upon binding to RNA and protein 32,33,[37][38][39] . For example, two short C-terminal helices of U1A provide an additional RNA interface for the interaction of its RRM domain with RNA 36,40 .…”

Section: Discussionmentioning

confidence: 95%

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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“…For the U1A RRM prototype, a similar α-helix occludes the RNP1 in the absence of RNA and rearranges in the RNA-bound conformation ( Fig. 3A; Oubridge et al 1994;Avis et al 1996). However, this RRM-helix association is likely to be relatively weak; for example, crystallization additives displace the α-helix from the RNP1 surface (Rupert et al 2003).…”

Section: Do Uhms Bind Rna?mentioning

confidence: 99%

Unmasking the U2AF homology motif family: a bona fide protein–protein interaction motif in disguise

Loerch

Kielkopf

2016

RNA

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U2AF homology motifs (UHM) that recognize U2AF ligand motifs (ULM) are an emerging family of protein-protein interaction modules. UHM-ULM interactions recur in pre-mRNA splicing factors including U2AF1 and SF3b1, which are frequently mutated in myelodysplastic syndromes. The core topology of the UHM resembles an RNA recognition motif and is often mistakenly classified within this large family. Here, we unmask the charade and review recent discoveries of UHM-ULM modules for protein-protein interactions. Diverse polypeptide extensions and selective phosphorylation of UHM and ULM family members offer new molecular mechanisms for the assembly of specific partners in the early-stage spliceosome.

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Solution Structure of the N-terminal RNP Domain of U1A Protein: The Role of C-terminal Residues in Structure Stability and RNA Binding

Cited by 167 publications

References 0 publications

Crystal structure of a core spliceosomal protein interface

Crystal structure of a core spliceosomal protein interface

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

Unmasking the U2AF homology motif family: a bona fide protein–protein interaction motif in disguise

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