Dataset Information

HnRNP L and hnRNP A1 induce extended U1 snRNA interactions with an exon to repress spliceosome assembly.

ABSTRACT: Pre-mRNA splicing is catalyzed through the activity of the spliceosome, a dynamic enzymatic complex. Forcing aberrant interactions within the spliceosome can reduce splicing efficiency and alter splice site choice; however, it is unknown whether such alterations are naturally exploited mechanisms of splicing regulation. Here, we demonstrate that hnRNP L represses CD45 exon 4 by recruiting hnRNP A1 to a sequence upstream of the 5' splice site. Together, hnRNP L and A1 induce extended contacts between the 5' splice site-bound U1 snRNA and neighboring exonic sequences that, in turn, inhibit stable association of U6 snRNA and subsequent catalysis. Importantly, analysis of several exons regulated by hnRNP L shows a clear relationship between the potential for binding of hnRNP A1 and U1 snRNA and the effect of hnRNP L on splicing. Together, our results demonstrate that conformational perturbations within the spliceosome are a naturally occurring and generalizable mechanism for controlling alternative splicing decisions.

SUBMITTER: Chiou NT

PROVIDER: S-EPMC3595347 | biostudies-literature | 2013 Mar

REPOSITORIES: biostudies-literature

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hnRNP L and hnRNP A1 induce extended U1 snRNA interactions with an exon to repress spliceosome assembly.

Chiou Ni-Ting NT Shankarling Ganesh G Lynch Kristen W KW

Molecular cell 20130207 5

Pre-mRNA splicing is catalyzed through the activity of the spliceosome, a dynamic enzymatic complex. Forcing aberrant interactions within the spliceosome can reduce splicing efficiency and alter splice site choice; however, it is unknown whether such alterations are naturally exploited mechanisms of splicing regulation. Here, we demonstrate that hnRNP L represses CD45 exon 4 by recruiting hnRNP A1 to a sequence upstream of the 5' splice site. Together, hnRNP L and A1 induce extended contacts bet ...[more]

PMID: 23394998

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Project description:7SK small nuclear RNA (snRNA) is an abundant and ubiquitously expressed noncoding RNA that functions to modulate the activity of RNA Polymerase II (RNAPII) in part by stabilizing distinct pools of 7SK-protein complexes. Prevailing models suggest that the secondary structure of 7SK is dynamically remodeled within its alternative RNA-protein pools such that its architecture differentially regulates the exchange of cognate binding partners. The nuclear hnRNP A1/A2 proteins influence the biology of 7SK snRNA via processes that require an intact stem loop (SL) 3 domain; however, the molecular details by which hnRNPs assemble onto 7SK snRNA are yet to be described. Here, we have taken an integrated approach to present a detailed description of the 7SK-hnRNP A1 complex. We show that unbound 7SK snRNA adopts at least two major conformations in solution, with significant structural differences localizing to the SL2-3 linker and the base of SL3. Phylogenetic analysis indicates that this same region is the least genetically conserved feature of 7SK snRNA. By performing DMS modifications with the presence of excess protein, we reveal that hnRNP A1 binds with selectivity to SL3 through mechanisms that increase the flexibility of the RNA adjacent to putative binding sites. Calorimetric titrations further validate that hnRNP A1-SL3 assembly is complex with the affinity of discrete binding events modulated by the surrounding RNA structure. To interpret this context-dependent binding phenomenon, we determined a 3D model of SL3 to show that it folds to position minimal hnRNP A1/A2 binding sites (5'-Y/RAG-3') within different local environments. SL3-protein complexes resolved by SEC-MALS-SAXS confirm that up to four hnRNP A1 proteins bind along the entire surface of SL3 via interactions that preserve the overall structural integrity of this domain. In sum, the collective results presented here reveal a specific role for a folded SL3 domain to scaffold hnRNP A1/A2-7SK assembly via mechanisms modulated by the surrounding RNA structure.

Dataset Information

HnRNP L and hnRNP A1 induce extended U1 snRNA interactions with an exon to repress spliceosome assembly.

Publications

hnRNP L and hnRNP A1 induce extended U1 snRNA interactions with an exon to repress spliceosome assembly.

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