Assembly of the U1 snRNP involves interactions with the backbone of the terminal stem of U1 snRNA

McConnell, Timothy S.; Lokken, R. Peter; Steitz, Joan A.

doi:10.1261/rna.2136103

Cited by 21 publications

(20 citation statements)

References 27 publications

(44 reference statements)

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“…Crystal and cryoelectron microscopy (cryo-EM) structures of purified or in vitro assembled U1 snRNPs show that SL4 is well separated from the first three stem-loops of U1 snRNA by the Sm ring (Stark et al 2001;Pomeranz Krummel et al 2009;Weber et al 2010). These as well as biochemical studies indicate that in the free snRNP, SL4 contacts the B and D2 subunits of the Sm core (McConnell et al 2003;Weber et al 2010). …”

Section: Discussionmentioning

confidence: 84%

“…The lack of interaction between the free snRNPs may indicate that a change in U1 conformation occurs upon binding the pre-mRNA that increases the accessibility of SL4. The contacts between SL4 and the Sm proteins B and D2 (McConnell et al 2003;Weber et al 2010) may prevent di-snRNP formation and then be altered upon U1 snRNP binding to the pre-mRNA. It will be interesting to examine the accessibility of these U1 nucleotides in different states of U1 snRNP assembly into the spliceosome.…”

Section: Discussionmentioning

confidence: 99%

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Stem–loop 4 of U1 snRNA is essential for splicing and interacts with the U2 snRNP-specific SF3A1 protein during spliceosome assembly

Sharma

Wongpalee

Vashisht³

et al. 2014

Genes Dev.

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The pairing of 59 and 39 splice sites across an intron is a critical step in spliceosome formation and its regulation. Interactions that bring the two splice sites together during spliceosome assembly must occur with a high degree of specificity and fidelity to allow expression of functional mRNAs and make particular alternative splicing choices. Here, we report a new interaction between stem-loop 4 (SL4) of the U1 snRNA, which recognizes the 59 splice site, and a component of the U2 small nuclear ribonucleoprotein particle (snRNP) complex, which assembles across the intron at the 39 splice site. Using a U1 snRNP complementation assay, we found that SL4 is essential for splicing in vivo. The addition of free U1-SL4 to a splicing reaction in vitro inhibits splicing and blocks complex assembly prior to formation of the prespliceosomal A complex, indicating a requirement for a SL4 contact in spliceosome assembly. To characterize the interactions of this RNA structure, we used a combination of stable isotope labeling by amino acids in cell culture (SILAC), biotin/Neutravidin affinity pull-down, and mass spectrometry. We show that U1-SL4 interacts with the SF3A1 protein of the U2 snRNP. We found that this interaction between the U1 snRNA and SF3A1 occurs within prespliceosomal complexes assembled on the pre-mRNA. Thus, SL4 of the U1 snRNA is important for splicing, and its interaction with SF3A1 mediates contact between the 59 and 39 splice site complexes within the assembling spliceosome.[Keywords: RNA-protein interaction; alternative splicing; gene expression; pre-mRNA splicing; ribonucleoprotein; snRNA; spliceosome] Supplemental material is available for this article. Intron removal is catalyzed by an ;40S RNP complex called the spliceosome, which consists of five small nuclear ribonucleoprotein particles (snRNPs) (U1, U2, U4, U5, and U6) and ;150 auxiliary proteins . In vitro, the spliceosome assembles onto an intron through the sequential binding of the snRNPs Hoskins et al. 2011). The 59 splice site is recognized by the U1 snRNP through base-pairing between the pre-mRNA and the snRNA. At the 39 end of the intron, the U2 auxiliary factor (U2AF) and splicing factor 1 (SF1) bind to the 39 splice site and branch point sequence, respectively. Initial association of the U2 snRNP can be ATP-independent and has been proposed to occur through interactions with the U2AF65 protein (Gozani et al. 1998;Das et al. 2000). This ATP-independent complex is referred to as the E complex. However, the stable association of U2 with the pre-mRNA requires ATP hydrolysis and formation of a base-pairing interaction between U2 snRNA and the branch point sequence with displacement of SF1. This pre-mRNP complex containing both the U1 and U2 snRNPs is called the prespliceosomal A complex. Recruitment of the U4/U6-U5 tri-snRNP forms the precatalytic B complex. In another mode of B complex formation, the U4/U6-U5 trisnRNP binds with the U2 snRNP to the 39 splice site, Ó 2014 Sharma et al. This article is distributed exclusively by Cold Spring...

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Section: Discussionmentioning

confidence: 84%

Section: Discussionmentioning

confidence: 99%

Stem–loop 4 of U1 snRNA is essential for splicing and interacts with the U2 snRNP-specific SF3A1 protein during spliceosome assembly

Sharma

Wongpalee

Vashisht³

et al. 2014

Genes Dev.

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“…The complex between the spliceosomal protein U1A and its target on the U1 small nuclear RNA has served as a model system for many studies. [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18] In this complex, the N-terminal RNA recognition motif of U1A interacts in a sequence-specific manner with stem/loop II (U1SLII) of the U1 small nuclear RNA ( Figure 1). Both experimental and computational studies have probed determinants of the binding affinity, and a number of experimental studies 1,7,9,10,[13][14][15] have been carried out to dissect the binding rate.…”

Section: Introductionmentioning

confidence: 99%

Prediction of Salt and Mutational Effects on the Association Rate of U1A Protein and U1 Small Nuclear RNA Stem/Loop II

Qin

Zhou

2007

J. Phys. Chem. B

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We have developed a computational approach for predicting protein-protein association rates (Alsallaq and Zhou, Structure 2007, 15, 215). Here we expand the range of applicability of this approach to protein-RNA binding and report the first results for protein-RNA binding rates predicted from atomistic modeling. The system studied is the U1A protein and stem/loop II of the U1 small nuclear RNA. Experimentally it was observed that the binding rate is significantly reduced by increasing salt concentration while the dissociation changes little with salt concentration, and charges distant from the binding site make marginal contribution to the binding rate. These observations are rationalized. Moreover, predicted effects of salt and charge mutations are found to be in quantitative agreement with experimental results.

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“…pT7U1, GFP-Spn, and GFP-SMN were cloned as described in refs. [16][17][18]. GFP-SMN* (siRNA target mutant) was generated by using the QuikChange PCR mutagenesis kit (Stratagene), along with 5Ј-AGA ACAGA ACT TA AGTGAC-CTACTTTCCCCAATCTGTGAAGTAGC-3Ј and 5Ј-GT-CACTTAAGTTCTGTTCTTCTCTATTTCCATATCCAGTG TAAAC-3Ј primers.…”

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confidence: 99%

Gemin proteins are required for efficient assembly of Sm-class ribonucleoproteins

Shpargel

Matera

2005

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

192

215

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Spinal muscular atrophy (SMA) is a neurodegenerative disease characterized by loss of spinal motor neurons. The gene encoding the survival of motor neurons (SMN) protein is mutated in >95% of SMA cases. SMN is the central component of a large oligomeric complex, including Gemins2-7, that is necessary and sufficient for the in vivo assembly of Sm proteins onto the small nuclear (sn)RNAs that mediate pre-mRNA splicing. After cytoplasmic assembly of the Sm core, both SMN and splicing snRNPs are imported into the nucleus, accumulating in Cajal bodies for additional snRNA maturation steps before targeting to splicing factor compartments known as ''speckles.'' In this study, we analyzed the function of individual SMN complex members by RNA interference (RNAi). RNAi-mediated knockdown of SMN, Gemin2, Gemin3, and Gemin4 each disrupted Sm core assembly, whereas knockdown of Gemin5 and Snurportin1 had no effect. Assembly activity was rescued by expression of a GFP-SMN construct that is refractive to RNAi but not by similar constructs that contain SMA patient-derived mutations. Our results also demonstrate that Cajal body homeostasis requires SMN and ongoing snRNP biogenesis. Perturbation of SMN function results in disassembly of Cajal bodies and relocalization of the marker protein, coilin, to nucleoli. Moreover, in SMN-deficient cells, newly synthesized SmB proteins fail to associate with U2 snRNA or accumulate in Cajal bodies. Collectively, our results identify a previously uncharacterized function for Gemin3 and Gemin4 in Sm core assembly and correlate the activity of this pathway with SMA.coilin ͉ DEAD box proteins ͉ RNA helicases ͉ small nuclear ribonucleoprotein biogenesis ͉ spinal muscular atrophy

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Assembly of the U1 snRNP involves interactions with the backbone of the terminal stem of U1 snRNA

Cited by 21 publications

References 27 publications

Stem–loop 4 of U1 snRNA is essential for splicing and interacts with the U2 snRNP-specific SF3A1 protein during spliceosome assembly

Stem–loop 4 of U1 snRNA is essential for splicing and interacts with the U2 snRNP-specific SF3A1 protein during spliceosome assembly

Prediction of Salt and Mutational Effects on the Association Rate of U1A Protein and U1 Small Nuclear RNA Stem/Loop II

Gemin proteins are required for efficient assembly of Sm-class ribonucleoproteins

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