Dynamic association of proteins with the pre-mRNA branch region.

MacMillan, Andrew M.; Query, Charles C.; Allerson, Charles R.; Chen, Swaine L.; Verdine, Gregory L.; Sharp, Phillip A.

doi:10.1101/gad.8.24.3008

Cited by 141 publications

(142 citation statements)

References 51 publications

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“…RNAs containing single [ 32 P] labels and/or photoactivatable cross-linking groups were constructed by splinted RNA-RNA ligations (Moore and Query 1998). Benzophenone-containing RNAs were prepared essentially as described (MacMillan et al 1994;Moore and Query 1998) by ligating the 10-nt RNA oligonucleotide (GGUGUCGCdA*G) containing a single convertible deoxyadenosine (dA*) to a series of [ 32 P]-phosphorylated 3Ј RNA transcripts derived from AdML-AG and AdML-GG, each containing progressively longer 5Ј exons. RNAs containing 4-thioU were constructed by initiating the 3Ј RNA fragment with synthetic 4-thioUpG during in vitro transcription.…”

Section: Rna Substrates and Antibodiesmentioning

confidence: 99%

5′ exon interactions within the human spliceosome establish a framework for exon junction complex structure and assembly

Reichert

Hir²,

Jurica³

et al. 2002

Genes Dev.

126

137

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A general consequence of pre-mRNA splicing is the stable deposition of several proteins 20-24 nucleotides (nt) upstream of exon-exon junctions on spliced mRNAs. This exon junction complex (EJC) contains factors involved in mRNA export, cytoplasmic localization, and nonsense-mediated mRNA decay. Here we probed the mechanism and timing of EJC assembly. Over the course of splicing, the 5 exon is subject to numerous dynamic protein-RNA interactions involving at least nine distinct polypeptides. Within the fully assembled spliceosome, these interactions afford protection to the last 25-27 nt of the 5 exon intermediate. Throughout their lifetimes in vivo, mRNAs exist as mRNA-protein particles (mRNPs). The associated proteins control every aspect of mRNA metabolism, from subcellular transport to translational efficiency to rate of decay. Exactly which proteins associate with a particular mRNA depends on its sequence, its subcellular localization, and its synthetic history. Furthermore, the complement of mRNP proteins evolves as the mRNA moves to different locations and is acted on by such processes as nuclear export and translation. Many proteins join the mRNP in the nucleus and then accompany it to the cytoplasm, where they influence subsequent mRNA metabolism ( These splicing-specific mRNP proteins constitute the exon junction complex (EJC), which associates with spliced mRNAs in a sequence-independent manner a fixed distance upstream of exon-exon junctions (Le Hir et al. 2000b). Using coimmunoprecipitation strategies, we and others recently reported that the EJC assembled in HeLa nuclear extract contains numerous epitopes, including those recognized by antibodies against REF/Aly, Y14, SRm160, DEK, RNPS1, UAP56, and Magoh (Blencowe et al. 1998;Mayeda et al. 1999;Kataoka et al. 2000Kataoka et al. , 2001Le Hir et al. 2000a,b, 2001bMcGarvey et al. 2000;Zhou et al. 2000;Gatfield et al. 2001;Luo et al. 2001). When assembled in vivo, the EJC is additionally precipitated by antibodies against Upf2, Upf3, and the TAP:p15 heterodimer (Kim et al. 2001b;Le Hir et al. 2001a;Lykke-Andersen et al. 2001). Finally, REF/Aly, Y14, SRm160, RNPS1, Upf2, Upf3, and TAP were all present in mRNPs immunopurified from the nuclear fraction of COS cells with antibodies against a subunit of the nuclear cap binding complex (Lejeune et al. 2002).The known EJC components function at multiple levels of mRNA metabolism. SRm160 and RNPS1 were originally characterized as activators of pre-mRNA splicing (Blencowe et al. 1998;Mayeda et al. 1999

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Section: Rna Substrates and Antibodiesmentioning

confidence: 99%

5′ exon interactions within the human spliceosome establish a framework for exon junction complex structure and assembly

Reichert

Hir²,

Jurica³

et al. 2002

Genes Dev.

126

137

View full text Add to dashboard Cite

show abstract

“…Previous studies in human and yeast extracts using hydroxy radical cleavage or protein-RNA crosslinking led to the hypothesis that the 5′SS and BS regions are closely positioned in early complexes containing only U1 and/or U2 (6)(7)(8)(9)(10)(11)(12). However, as both of these irreversible trapping methods can capture transient excursions that are not necessarily on the pathway for splicing, when during spliceosome assembly and activation the 5′SS and BS are stably juxtaposed has remained unclear.…”

mentioning

confidence: 99%

Single-molecule colocalization FRET evidence that spliceosome activation precedes stable approach of 5′ splice site and branch site

Crawford

Hoskins

Friedman

et al. 2013

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

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Removal of introns from the precursors to messenger RNA (premRNAs) requires close apposition of intron ends by the spliceosome, but when and how apposition occurs is unclear. We investigated the process by which intron ends are brought together using single-molecule fluorescence resonance energy transfer together with colocalization single-molecule spectroscopy, a combination of methods that can directly reveal how conformational transitions in macromolecular machines are coupled to specific assembly and disassembly events. The FRET measurements suggest that the 5′ splice site and branch site remain physically separated throughout spliceosome assembly, and only approach one another after the spliceosome is activated for catalysis, at which time the pre-mRNA becomes highly dynamic. Separation of the sites of chemistry until very late in the splicing pathway may be crucial for preventing splicing at incorrect sites.splicing mechanism | single-molecule FRET | RNA dynamics I ntron excision from precursors to messenger RNAs (premRNAs) is carried out by the spliceosome, arguably the most complex macromolecular machine in the cell (1). One of the most important jobs of the spliceosome is to accurately and efficiently identify the ends of introns and bring them together to promote the chemistry of splicing. This chemistry occurs via two S N 2 transesterification reactions: (i) attack by the branch site (BS) adenosine on the phosphodiester bond at the beginning of the intron (the 5′ splice site; 5′SS) and (ii) attack of the released 5′ exon on the phosphodiester bond at the end of the intron (the 3′SS) (2). The BS adenosine is internal to the intron and usually located in the vicinity of the 3′SS.The spliceosome consists of four major subcomplexes that must assemble de novo on each new intron: the U1 and U2 small nuclear ribonucleoprotein particles (snRNPs), the U4/U6.U5 tri-snRNP, and the protein-only nineteen complex (NTC). The snRNPs each contain numerous proteins and one or more small nuclear RNAs (snRNAs). These subcomplexes assemble stepwise, with U1 and U2 recognition of the 5′SS and BS, respectively, preceding trisnRNP and NTC recruitment (3, 4). Throughout the assembly process, numerous large-scale conformational changes occur that involve making and breaking of pre-mRNA:snRNA and snRNA: snRNA base pairing interactions. These structural transitions are necessary for both recognition of the splice sites and creation of the catalytic core in which the splice sites are juxtaposed for chemistry. The two chemical steps occur within the activated spliceosome formed after ejection of the U1 and U4 snRNPs (5).When during spliceosome assembly are the splice sites brought into close proximity? Previous studies in human and yeast extracts using hydroxy radical cleavage or protein-RNA crosslinking led to the hypothesis that the 5′SS and BS regions are closely positioned in early complexes containing only U1 and/or U2 (6-12). However, as both of these irreversible trapping methods can capture transient excursions that are no...

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“…Site-specific modification of the pre-mRNA branch adenosine with benzophenone followed by photocrosslinking revealed the specific, sequential association of several factors with the branch adenosine from very early in spliceosome assembly (6). Of particular interest was a strong crosslink to p14 that appeared in A complex and persisted within the fully assembled spliceosome.…”

mentioning

confidence: 99%

“…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.…”

mentioning

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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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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Dynamic association of proteins with the pre-mRNA branch region.

Cited by 141 publications

References 51 publications

5′ exon interactions within the human spliceosome establish a framework for exon junction complex structure and assembly

5′ exon interactions within the human spliceosome establish a framework for exon junction complex structure and assembly

Single-molecule colocalization FRET evidence that spliceosome activation precedes stable approach of 5′ splice site and branch site

Crystal structure of a core spliceosomal protein interface

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