The human Prp8 protein is a component of both U2- and U12-dependent spliceosomes

Luo, Hongbo; Moreau, G.; Levin, Natasha; Moore, Melissa J.

doi:10.1017/s1355838299990520

Cited by 89 publications

(72 citation statements)

References 62 publications

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“…The conserved sequences in the ribozyme and the U6 snRNA could have a common function but the results summarized above are far from being conclusive+ Recent evidence indicates that the protein Prp8 is also closely associated in the spliceosome with the 59 splice site, branch site, and 39 splice site (Collins & Guthrie, 1999;Luo et al+, 1999;Reyes et al+, 1999;Siatecka et al+, 1999)+ It is possible that this or another protein could be a component of the active site for the first step in splicing, perhaps in conjunction with U2 and U6 snRNA+ These possibilities are difficult to investigate because the conformations of snRNAs change during the splicing process and the heart of the spliceosome is relatively inaccessible to probes+ In the near term, it will be important to determine the structure of the 29-59AG1 ribozyme to gain insight into the specific function of the ACAGAGA sequence in lariat formation+ It may then be possible to test if the analogous residues in U6 snRNA have a related function in the spliceosome+…”

Section: Discussionmentioning

confidence: 99%

A ribozyme selected from variants of U6 snRNA promotes 2′,5′-branch formation

Tuschl

Sharp

Bartel

2001

RNA

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In vitro selection was used to sample snRNA-related sequences for ribozyme activities, and several 29,59-branchforming ribozymes were isolated. One such ribozyme is highly dependent upon an 11-nt motif that contains a conserved U6 snRNA sequence (ACAGAGA-box) known to be important for pre-mRNA splicing. The ribozyme reaction is similar to the first step of splicing in that an internal 29-hydroxyl of an unpaired adenosine attacks at the 59-phosphate of a guanosine. It differs in that the leaving group is diphosphate rather than a 59 exon. The finding that lariat formation can be accomplished by a small RNA with sequences related to U6 snRNA indicates that the RNA available in the spliceosome may be involved in RNA-catalyzed branch formation.

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Section: Discussionmentioning

confidence: 99%

A ribozyme selected from variants of U6 snRNA promotes 2′,5′-branch formation

Tuschl

Sharp

Bartel

2001

RNA

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“…The few essential splicing factors that have been examined in vertebrates show surprisingly variable levels of expression among different tissues that do not correlate with tissue metabolic activity. For example, SF1, a spliceosome component involved in the initial recognition of the branch site, is barely detectable in pancreas, kidney, and lung, whereas PRP8 is barely detectable in liver (Luo et al 1999;Vervoort et al 2000).…”

Section: What Is the Basis For Cell-specificity In Rp And Sma?mentioning

confidence: 99%

Pre-mRNA splicing and human disease

Faustino¹,

Cooper²

2003

Genes Dev.

1,142

862

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The precision and complexity of intron removal during pre-mRNA splicing still amazes even 26 years after the discovery that the coding information of metazoan genes is interrupted by introns (Berget et al. 1977;Chow et al. 1977). Adding to this amazement is the recent realization that most human genes express more than one mRNA by alternative splicing, a process by which functionally diverse protein isoforms can be expressed according to different regulatory programs. Given that the vast majority of human genes contain introns and that most pre-mRNAs undergo alternative splicing, it is not surprising that disruption of normal splicing patterns can cause or modify human disease. The purpose of this review is to highlight the different mechanisms by which disruption of pre-mRNA splicing play a role in human disease. Several excellent reviews provide detailed information on splicing and the regulation of splicing (Burge et al. 1999;Hastings and Krainer 2001; Black 2003). The potential role of splicing as a modifier of human disease has also recently been reviewed (NissimRafinia and Kerem 2002). Constitutive splicing and the basal splicing machineryThe typical human gene contains an average of 8 exons. Internal exons average 145 nucleotides (nt) in length, and introns average more than 10 times this size and can be much larger (Lander et al. 2001). Exons are defined by rather short and degenerate classical splice-site sequences at the intron/exon borders (5Ј splice site, 3Ј splice site, and branch site; Fig. 1A). Components of the basal splicing machinery bind to the classical splice-site sequences and promote assembly of the multicomponent splicing complex known as the spliceosome. The spliceosome performs the two primary functions of splicing: recognition of the intron/exon boundaries and catalysis of the cut-and-paste reactions that remove introns and join exons. The spliceosome is made up of five small nuclear ribonucleoproteins (snRNPs) and >100 proteins. Each snRNP is composed of a single uridinerich small nuclear RNA (snRNA) and multiple proteins. The U1 snRNP binds the 5Ј splice site, and the U2 snRNP binds the branch site via RNA:RNA interactions between the snRNA and the pre-mRNA (Fig. 1B). Spliceosome assembly is highly dynamic in that complex rearrangements of RNA:RNA, RNA:protein, and protein:protein interactions take place within the spliceosome. Coinciding with these internal rearrangements, both splice sites are recognized multiple times by interactions with different components during the course of spliceosome assembly (for example, see Burge et al. 1999;Du and Rosbash 2002;Lallena et al. 2002;Liu 2002). The catalytic component is likely to be U6 snRNP, which joins the spliceosome as a U4/U6 · U5 tri-snRNP (Villa et al. 2002).A splicing error that adds or removes even 1 nt will disrupt the open reading frame of an mRNA; yet exons are correctly spliced from within tens of thousands of intronic nucleotides. This remarkable precision is, in part, built into the mechanism of intron removal because once...

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“…The highly conserved U5-associated protein, U5 220-kDa (Prp8), important in both tri-snRNP formation and spliceosome assembly (Brown & Beggs, 1992), is currently the most likely protein to participate directly in catalysis+ Prp8 has been implicated in interactions with both 59 and 39 splice sites, and the polypyrimidine tract (Teigelkamp et al+, 1995a(Teigelkamp et al+, , 1995bUmen & Guthrie, 1995;Reyes et al+, 1996)+ Mutations in Prp8 have been shown to suppress mutations at the 59 and 39 splice sites, as well as mutations in the U6 ACAGAGA box and U4 snRNA (Collins & Guthrie, 1999;Kuhn et al+, 1999;Siatecka et al+, 1999), which may suggest a role for Prp8 as a general stabilizing element for RNA-RNA interactions in the spliceosome+ Precedents for such roles for proteins in RNA-centric catalytic systems have been demonstrated for group I introns, RNase P, and the hammerhead ribozyme (Guerrier-Takada et al+, 1983;Tsuchihashi et al+, 1993;Weeks & Cech, 1995)+ The fact that U5, and Prp8, are involved in splicing of both U2-dependent and U12-dependent introns indicates that the function of Prp8 does not depend on the identity of nucleotides at the splice junctions (Tarn & Steitz, 1996;Luo et al+, 1999)+ Alternatively, the apparent lack of specificity might result from redundancy with another spliceosomal component; for example, the function of loop I of U5 can be fulfilled by other U5-associated components, probably Prp8, which in fact has an interaction pattern with splice sites similar to that of loop I (O'Keefe et al+, 1996;Segault et al+, 1999)+…”

Section: The Long Range U2-u6 Interaction Is Not Disrupted By Mutatiomentioning

confidence: 99%

A tertiary interaction detected in a human U2-U6 snRNA complex assembled in vitro resembles a genetically proven interaction in yeast

Valadkhan

Manley

2000

RNA

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U2 and U6 small nuclear RNAs are thought to play critical roles in pre-mRNA splicing catalysis. Genetic evidence suggests they form an extensively base-paired structure within the spliceosome that is required for catalysis. Especially in light of significant similarities with group II self-splicing introns, we wished to investigate whether the purified RNAs might by themselves be able to form a complex similar to that which appears to exist in the spliceosome. To this end, we synthesized and purified large segments of human U2 and U6 snRNAs. Upon annealing, the two RNAs efficiently formed a stable and apparently extensively base-paired (T m 5 50-60 8C in the presence of 20 mM Mg 21 ) complex. To investigate possible tertiary interactions, we subjected the annealed complex to UV irradiation, and two crosslinked species were identified and characterized. The major one links the second G in the highly conserved and critical ACAGAGA sequence in U6 with an A in U2 just 59 to U2-U6 helix Ia and opposite the invariant AGC in U6. Remarkably, this crosslink indicates a tertiary interaction essentially identical to one detected previously by genetic covariation in yeast. Together our results suggest that purified U2 and U6 snRNAs can anneal and fold to form a structure resembling that likely to exist in the catalytically active spliceosome.

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The human Prp8 protein is a component of both U2- and U12-dependent spliceosomes

Cited by 89 publications

References 62 publications

A ribozyme selected from variants of U6 snRNA promotes 2′,5′-branch formation

A ribozyme selected from variants of U6 snRNA promotes 2′,5′-branch formation

Pre-mRNA splicing and human disease

A tertiary interaction detected in a human U2-U6 snRNA complex assembled in vitro resembles a genetically proven interaction in yeast

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