Efficient association of U2 snRNPs with pre-mRNA requires an essential U2 RNA structural element.

Zavanelli, M I; Ares, Manuel

doi:10.1101/gad.5.12b.2521

Cited by 65 publications

(115 citation statements)

References 62 publications

Supporting

Mentioning

110

Contrasting

Order By: Relevance

“…The alternative conformation is likely to be biologically significant because the potential for alternative base pairing is conserved from yeast to humans (74). Yet, despite prodigious effort (39,49,59), there has been no direct evidence that the alternative U2 conformation is required for, or occurs during, the normal mRNA splicing reaction. Judging by increased reactivity of U65, U2 loop IIa may be partially exposed in the aberrant U2 snRNP conformation brought about by trans-spliceosome assembly on the non-functional UP-GG mutant (Fig.…”

Section: Discussionmentioning

confidence: 99%

“…Intriguingly, SF3 proteins in complex A can also bind to a 20 nt region of the mRNA precursor, known as the anchoring site, that is located just upstream of the BS (18). Moreover, SF3 components can functionally suppress U2 stem-loop IIa mutations in yeast (49,59,61,62). Thus, SF3b proteins might interact simultaneously with U2 snRNA stem-loop IIa and with the anchoring site on the mRNA precursor; alternatively, SF3b might be partially transferred from U2 snRNA to the anchoring site on the mRNA precursor (Fig.…”

Section: Discussionmentioning

confidence: 99%

“…2A and B) and induce U2/U6 helix II formation (Fig. 4); (ii) a region within the upstream sequences called the anchoring site is bound in complex A by intrinsic U2 snRNP proteins known as SF3 (18); and (iii) yeast SF3 proteins can functionally suppress mutations in U2 snRNA stem-loop IIa (49,59,61,62).…”

Section: Non-functional Upstream Sequences Induce a Conformational Chmentioning

confidence: 99%

“…In addition, because the U2 snRNP-associated SF3 proteins interact with the anchoring site (18), we were also intrigued by the observation that SF3 proteins (59) can suppress mutations in U2 stem-loop IIa, suggesting that SF3 proteins and/or the U2 snRNP-associated CUS2 protein (60) could mediate a U2 conformational change between alternative phylogenetically conserved structures (49,(59)(60)(61)(62). Consistent with these earlier observations, we show here that (i) functional sequences upstream of the branch site are required for stable U2/BS association in complex A and for formation of U2/U6 helix II; and (ii) non-functional upstream sequences not only fail to support stable assembly of complex A, but cause misfolding of U2 stem-loop IIa.…”

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Sequences upstream of the branch site are required to form helix II between U2 and U6 snRNA in a trans-splicing reaction

Ast

Pavelitz

Weiner

2001

Nucleic Acids Research

View full text Add to dashboard Cite

Three different base paired stems form between U2 and U6 snRNA over the course of the mRNA splicing reaction (helices I, II and III). One possible function of U2/U6 helix II is to facilitate subsequent U2/U6 helix I and III interactions, which participate directly in catalysis. Using an in vitro trans-splicing assay, we investigated the function of sequences located just upstream from the branch site (BS). We find that these upstream sequences are essential for stable binding of U2 to the branch region, and for U2/U6 helix II formation, but not for initial U2/BS pairing. We also show that non-functional upstream sequences cause U2 snRNA stem-loop IIa to be exposed to dimethylsulfate modification, perhaps reflecting a U2 snRNA conformational change and/or loss of SF3b proteins. Our data suggest that initial binding of U2 snRNP to the BS region must be stabilized by an interaction with upstream sequences before U2/U6 helix II can form or U2 stem-loop IIa can participate in spliceosome assembly.

show abstract

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Non-functional Upstream Sequences Induce a Conformational Chmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Sequences upstream of the branch site are required to form helix II between U2 and U6 snRNA in a trans-splicing reaction

Ast

Pavelitz

Weiner

2001

Nucleic Acids Research

View full text Add to dashboard Cite

show abstract

“…Some biochemical evidence suggests that SF3B2 may directly contact stem IIa (Kramer et al 1999), and genetic interactions point to a functional relationship (Wells et al 1996;Yan and Ares 1996). During prespliceosome assembly, the stem IIa conformation is required for stable U2 snRNP association with pre-mRNA and branch point recognition (Zavanelli and Ares 1991;Perriman and Ares 2007). Both of these rearrangements require the RNA-dependent ATPase Prp5.…”

Section: Discussionmentioning

confidence: 99%

Rearrangements within human spliceosomes captured after exon ligation

Ilagan

Chalkley

Burlingame

et al. 2013

RNA

View full text Add to dashboard Cite

In spliceosomes, dynamic RNA/RNA and RNA/protein interactions position the pre-mRNA substrate for the two chemical steps of splicing. Not all of these interactions have been characterized, in part because it has not been possible to arrest the complex at clearly defined states relative to chemistry. Previously, it was shown in yeast that the DEAD/H-box protein Prp22 requires an extended 3 ′ exon to promote mRNA release from the spliceosome following second-step chemistry. In line with that observation, we find that shortening the 3 ′ exon blocks cleaved lariat intron and mRNA release in human splicing extracts, which allowed us to stall human spliceosomes in a new post-catalytic complex (P complex). In comparison to C complex, which is blocked at a point following first-step chemistry, we detect specific differences in RNA substrate interactions near the splice sites. These differences include extended protection across the exon junction and changes in protein crosslinks to specific sites in the 5 ′ and 3 ′ exons. Using selective reaction monitoring (SRM) mass spectrometry, we quantitatively compared P and C complex proteins and observed enrichment of SF3b components and loss of the putative RNA-dependent ATPase DHX35. Electron microscopy revealed similar structural features for both complexes. Notably, additional density is present when complexes are chemically fixed, which reconciles our results with previously reported C complex structures. Our ability to compare human spliceosomes before and after second-step chemistry has opened a new window to rearrangements near the active site of spliceosomes, which may play roles in exon ligation and mRNA release.

show abstract

In vitro reconstitution of mammalian U2 and U5 snRNPs active in splicing: Sm proteins are functionally interchangeable and are essential for the formation of functional U2 and U5 snRNPs.

Ségault¹,

Will²,

Sproat³

et al. 1995

The EMBO Journal

View full text Add to dashboard Cite

An in vitro reconstitution/splicing complementation system has been developed which has allowed the investigation of the role of mammalian U2 and U5 snRNP components in splicing. U2 or U5 snRNP cores are first reconstituted from purified native snRNP core proteins and snRNA in the absence of cellular extract and are subsequently added to splicing extracts depleted of either U2 or U5 snRNP. When snRNPs reconstituted with HeLa U2 or U5 snRNA were added to U2‐ or U5‐depleted nuclear extract, splicing was complemented. Addition of naked snRNA, on the other hand, did not restore splicing, demonstrating that the core proteins are essential for both U2 and U5 snRNP functions in splicing. Hybrid U2 or U5 snRNPs, reconstituted with core proteins isolated from U1 or U2 snRNPs, were equally active in splicing complementation, indicating that the snRNP core proteins are functionally interchangeable. U5 snRNPs reconstituted from in vitro transcribed U5 snRNA restored splicing to a level identical to that observed with particles reconstituted from authentic HeLa U5 snRNA. In contrast, splicing could not be restored to U2‐depleted extract by the addition of snRNPs reconstituted from synthetic U2 snRNA, suggesting that U2 snRNA base modifications are essential for U2 snRNP function.

show abstract

Efficient association of U2 snRNPs with pre-mRNA requires an essential U2 RNA structural element.

Cited by 65 publications

References 62 publications

Sequences upstream of the branch site are required to form helix II between U2 and U6 snRNA in a trans-splicing reaction

Sequences upstream of the branch site are required to form helix II between U2 and U6 snRNA in a trans-splicing reaction

Rearrangements within human spliceosomes captured after exon ligation

In vitro reconstitution of mammalian U2 and U5 snRNPs active in splicing: Sm proteins are functionally interchangeable and are essential for the formation of functional U2 and U5 snRNPs.

Contact Info

Product

Resources

About