In vitro assembly of U1 snRNPs.

Hamm, Jörg; Kazmaier, Michaël; Mattaj, Iain W.

doi:10.1002/j.1460-2075.1987.tb02672.x

Cited by 147 publications

(137 citation statements)

References 36 publications

(26 reference statements)

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“…U6 was present, but at less than 500-fold coverage, because of its lack of a TMG cap. The remaining RNAs were examined for the expected hallmarks of U1 snRNA: complementarity to known 5′ splice sites in C. merolae (GUAAGU) (6), presence of an Sm-binding site, and binding sites for conserved U1 proteins U1-A and U1-70K (28). Eleven RNAs had a potential Sm-binding site and complementarity to the 5′ splice site, and five of these also contained one canonical U1 proteinbinding site, but sequence alignments to U1 sequences from the Rfam database (29) failed to reveal significant sequence similarity.…”

Section: Resultsmentioning

confidence: 99%

Dramatically reduced spliceosome in Cyanidioschyzon merolae

Stark

Dunn²,

Dunn

et al. 2015

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

143

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The human spliceosome is a large ribonucleoprotein complex that catalyzes pre-mRNA splicing. It consists of five snRNAs and more than 200 proteins. Because of this complexity, much work has focused on the Saccharomyces cerevisiae spliceosome, viewed as a highly simplified system with fewer than half as many splicing factors as humans. Nevertheless, it has been difficult to ascribe a mechanistic function to individual splicing factors or even to discern which are critical for catalyzing the splicing reaction. We have identified and characterized the splicing machinery from the red alga Cyanidioschyzon merolae, which has been reported to harbor only 26 intron-containing genes. The U2, U4, U5, and U6 snRNAs contain expected conserved sequences and have the ability to adopt secondary structures and form intermolecular base-pairing interactions, as in other organisms. C. merolae has a highly reduced set of 43 identifiable core splicing proteins, compared with ∼90 in budding yeast and ∼140 in humans. Strikingly, we have been unable to find a U1 snRNA candidate or any predicted U1-associated proteins, suggesting that splicing in C. merolae may occur without the U1 small nuclear ribonucleoprotein particle. In addition, based on mapping the identified proteins onto the known splicing cycle, we propose that there is far less compositional variability during splicing in C. merolae than in other organisms. The observed reduction in splicing factors is consistent with the elimination of spliceosomal components that play a peripheral or modulatory role in splicing, presumably retaining those with a more central role in organization and catalysis.pre-mRNA splicing | spliceosome core | U1 snRNP | genome reduction | splicing mechanism P re-mRNA splicing occurs by two transesterification reactions that are catalyzed by the spliceosome, a large macromolecular assembly of five snRNAs and more than 200 proteins in humans (1). These components are thought to assemble onto each new pre-mRNA transcript in an ordered fashion through the recognition and binding of three highly conserved sequences in the transcript: the 5′ splice site, the branch site, and the 3′ splice site (2, 3). Some of these interactions occur via direct RNA/RNA base pairing between the transcript and snRNAs; for example, both U1 and U6 snRNAs base pair to the 5′ splice site of the pre-mRNA transcript, and, similarly, U2 snRNA base pairs to the branch site (3).Given the complexity of the human spliceosome, it is of considerable interest to find a more tractable splicing system with fewer components to study the core processes of splicing (assembly, catalysis, and fidelity). The Saccharomyces cerevisiae (yeast) spliceosome has been proposed as a simplified model system, because it contains only about 100 proteins (4). Indeed, substantial progress in understanding the spliceosome has been made by studying yeast splicing (3,5). Nevertheless, the yeast spliceosome is still a highly complex system in which to investigate the role of individual proteins, let alone attempt ...

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Section: Resultsmentioning

confidence: 99%

Dramatically reduced spliceosome in Cyanidioschyzon merolae

Stark

Dunn²,

Dunn

et al. 2015

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

143

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“…protein makes contacts with Ul snRNA at the first stem-loop (20,41) in a sequence-specific manner (46,56). The carboxyterminal portion of the protein is particularly rich in arginine residues and contains repetitions of arginine-glutamic acid, arginine-aspartic acid, and arginine-serine.…”

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confidence: 99%

Structure and expression of the Drosophila melanogaster gene for the U1 small nuclear ribonucleoprotein particle 70K protein.

Mancebo¹,

Lo²,

Mount³

1990

Mol. Cell. Biol.

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A genomic clone encoding the Drosophila Ul small nuclear ribonucleoprotein particle 70K protein was isolated by hybridization with a human Ul small nuclear ribonucleoprotein particle 70K protein cDNA. Southern blot and in situ hybridizations showed that this Ul 70K gene is unique in the Drosophila genome, residing at cytological position 27D1,2. Polyadenylated transcripts of 1.9 and 3.1 kilobases were observed. While the 1.9-kilobase mRNA is always more abundant, the ratio of these two transcripts is developmentally regulated. Analysis of cDNA and genomic sequences indicated that these two RNAs encode an identical protein with a predicted molecular weight of 52,879. Comparison of the Ul 70K proteins predicted from Drosophila, human, and Xenopus cDNAs revealed 68% amino acid identity in the most amino-terminal 214 amino acids, which include a sequence motif common to many proteins which bind RNA. The carboxy-terminal half is less well conserved but is highly charged and contains distinctive arginine-rich regions in all three species. These arginine-rich regions contain stretches of arginine-serine dipeptides like those found in transformer, transformer-2, and suppressor-of-white-apricot proteins, all of which have been identified as regulators of mRNA splicing in Drosophila melanogaster.Splicing of pre-mRNA occurs in a two-step reaction requiring the assembly of a large complex (the spliceosome) which includes several small nuclear ribonucleoprotein particles (snRNPs) (see references 19, 32, and 52 for reviews). Each snRNP consists of at least one snRNA bound to several proteins, some of which are common to all the spliceosomal snRNPs. The human Ul snRNP is composed of a 164-nucleotide (nt) Ul snRNA molecule complexed to at least 10 proteins (30). It binds to 5' splice site sequences by base pairs involving the 5' end of Ul RNA (12,24,35,62). There is also evidence that the Ul snRNP interacts with the U2 snRNP during splicing (8,12), and that this interaction occurs early and is required for further stages of splicing (25,48,50). Three of the human Ul snRNP proteins, 70K, A, and C, are unique to the Ul snRNP (21, 43); the other Ul snRNP proteins are common to the U2, U5, and U4/U6 snRNPs.Patients with autoimmune disorders produce antibodies which specifically immunoprecipitate the Ul snRNP (28), and many of these are directed against epitopes on the 70K protein (43, 60). Anti-RNP antisera have been shown to block in vitro splicing when they are added to splicing reactions (40). Such sera, as well as a mouse monoclonal antibody to the mouse Ul snRNP 70K protein (6), have enabled researchers to clone cDNAs encoding the Ul 70K protein from expression libraries (47,59).Sequence data from these cDNAs have provided information regarding the primary structure of the Ul 70K protein.First, it appears that this protein is actually 52 kilodaltons in size (45, 53). We have followed others (15, 46) in using Ul 70K or 70K as a name for this protein, although this name says nothing about the size of the protein. A conserved reg...

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“…Previous studies of the RNA-binding properties of the Ul snRNP-specific proteins have involved indirect methods including immunoprecipitation of nuclease-treated cell extracts (34) and microinjection of RNA into Xenopus oocytes (12). These studies suggested that the 70K protein, as well as the A protein and perhaps the C protein, associates with the 5' half of Ul RNA, in particular, stem-loop I.…”

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A specific 31-nucleotide domain of U1 RNA directly interacts with the 70K small nuclear ribonucleoprotein component.

1989

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We have defined the nucleotide sequence of a protein-binding domain within Ul RNA that specifically recognizes and binds both to a Ul small nuclear ribonucleoprotein component (the 70K protein) and to the previously defined RNA-binding domain of the 70K protein. We have investigated direct interactions between purified Ul RNA and 70K protein by reconstitution in vitro. Thirty-one nucleotides of Ul RNA, corresponding to stem-loop I, were required for this interaction. Nucleotides at the 5' end of Ul RNA that are involved in base pairing with the 5' splice site of pre-mRNA were not required for binding. In contrast to other reports, these findings demonstrate that a specific domain of Ul RNA can bind directly to the 70K protein independently of any other snRNP-associated proteins.The 52-kilodalton Ul RNA-associated protein (the 70K protein) is a component of the Ul small nuclear ribonucleoprotein (snRNP) complex. Several snRNPs have been implicated in the removal of intervening sequences during precursor mRNA splicing (see references 11, 25, and 41 for reviews). The Ul snRNP, in particular, has been shown to interact with the 5' splice site, at least in part by base pairing of this site with the 5'-terminal 10 nucleotides (nt) of Ul RNA (1,5,18,29,45). The human Ul snRNP is composed of 165 nt of Ul RNA and two classes of proteins: Ul-specific proteins (70K, A, and C) and the Sm complex (consisting of six U snRNP-common proteins) (9, 22). Although these proteins probably play important structural roles in snRNPs and at least some are required for the interaction of Ul snRNP with 5' splice sites (29), none have been assigned specific functions.Previous studies of the RNA-binding properties of the Ul snRNP-specific proteins have involved indirect methods including immunoprecipitation of nuclease-treated cell extracts (34) and microinjection of RNA into Xenopus oocytes (12). These studies suggested that the 70K protein, as well as the A protein and perhaps the C protein, associates with the 5' half of Ul RNA, in particular, stem-loop I. In addition, weak requirements were implicated for regions of stemloops II, III, and IV (12,34 purified human Ul RNA and 70K protein, we have investigated the direct interaction of these two components of the Ul snRNP. Using various binding methods, we maintained the specificity of the Ul RNA-70K reconstitution through progressive deletion of both components. We previously defined the RNA-binding domain of the 70K protein required for this interaction (35). In this study, we report that both the 70K protein and the RNA-binding domain alone are directly recognized by 31 nt of Ul RNA that make up the 5'-most stem-loop structure. The A, C, and Sm proteins were not required for this recognition and binding. Further deletion of the 31-nt structure resulted in reduced efficiency of reconstitution, thus defining the minimal RNA domain needed for recognition of the 70K protein. MATERIALS AND METHODS

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In vitro assembly of U1 snRNPs.

Cited by 147 publications

References 36 publications

Dramatically reduced spliceosome in Cyanidioschyzon merolae

Dramatically reduced spliceosome in Cyanidioschyzon merolae

Structure and expression of the Drosophila melanogaster gene for the U1 small nuclear ribonucleoprotein particle 70K protein.

A specific 31-nucleotide domain of U1 RNA directly interacts with the 70K small nuclear ribonucleoprotein component.

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