Conserved Loop I of U5 Small Nuclear RNA Is Dispensable for Both Catalytic Steps of Pre-mRNA Splicing in HeLa Nuclear Extracts

Abstract

The function of conserved regions of the metazoan U5 snRNA was investigated by reconstituting U5 small nuclear ribonucleoprotein particles (snRNPs) from purified snRNP proteins and HeLa or Xenopus U5 snRNA mutants and testing their ability to restore splicing to U5-depleted nuclear extracts. Substitution of conserved nucleotides comprising internal loop 2 or deletion of internal loop 1 had no significant effect on the ability of reconstituted U5 snRNPs to complement splicing. However, deletion of internal loop 2 abolished U5 activity in splicing and spliceosome formation. Surprisingly, substitution of the invariant loop 1 nucleotides with a GAGA tetraloop had no effect on U5 activity. Furthermore, U5 snRNPs reconstituted from an RNA formed by annealing the 5 and 3 halves of the U5 snRNA, which lacked all loop 1 nucleotides, complemented both steps of splicing. Thus, in contrast to yeast, loop 1 of the human U5 snRNA is dispensable for both steps of splicing in HeLa nuclear extracts. This suggests that its function can be compensated for in vitro by other spliceosomal components: for example, by proteins associated with the U5 snRNP. Consistent with this idea, immunoprecipitation studies indicated that several functionally important U5 proteins associate stably with U5 snRNPs containing a GAGA loop 1 substitution.Nuclear pre-mRNA splicing proceeds via a two-step mechanism. In the first step, the pre-mRNA is hydrolyzed at the 5Ј splice site and the 5Ј end of the intron interacts concomitantly with an adenosine at the so-called branch point. The splicing intermediates thus generated include exon 1 and a lariat structure comprised of the intron and exon 2. In the second step, hydrolysis at the 3Ј splice site and the concomitant ligation of exons 1 and 2 give rise to the mRNA and the excised intron in the form of a lariat. Both reactions are catalyzed by the spliceosome, a large ribonucleoprotein complex formed by the ordered interaction of numerous splicing factors and the four small nuclear ribonucleoprotein particles (snRNPs), U1, U2, U5, and U4/U6, with conserved regions of the pre-mRNA (reviewed in references 19, 27, and 34). Spliceosome assembly is initiated by the interaction of the U1 and U2 snRNPs with the 5Ј splice site and branch site, respectively, thereby generating the so-called prespliceosome, or complex A. Mature spliceosomes (i.e., complexes B and C) are ultimately formed by the subsequent interaction of the U4/U6 and U5 snRNPs, in the form of a preassembled U4/U6.U5 tri-snRNP complex (reviewed in references 19 and 34).The assembly of a catalytically active spliceosome requires the formation of a network of RNA-RNA interactions which favorably position the chemically reactive groups of the premRNA for catalysis (for reviews, see references 26 and 38). The U5 snRNP has been proposed to play a central role in recognizing and aligning the 5Ј and 3Ј splice sites for catalysis, and its function appears to be mediated, at least in part, by base pairing interactions between the U5 small nuclear RNA (snRNA) ...

PSF and p54nrb bind a conserved stem in U5 snRNA

“…weakly, but such interaction might be stabilized by the presence of PSF+ Binding specificity for both proteins appears to depend on multiple elements, including the predicted binding sequence on the 39 side of stem 1b+ The importance of the 39 strand of the stem 1b was first identified in SELEX assays and confirmed using the 59 and 39 mutant U5 snRNAs+ In both the filter-binding assays and the biotinylated-RNA selection experiments, PSF and p54 nrb bound to wild-type U5 and the 59 mutant RNA more efficiently than to the 39 mutant+ However, the importance of the stem structure was shown using the 59-39 double mutant+ Because the 59-39 mutant lacks the optimal binding sequence for PSF-p54 nrb , it seems that the structure of stem 1b contributes to binding specificity as well+ Although we have not experimentally verified whether the 59-39 mutant adopts a structure identical to wild-type U5, computer folding algorithms predict the same secondary structure+ Whether other factors also contribute to binding specificity remains unclear+ Secondary structure predictions of the 59 and 39 mutants suggest that these mutations primarily affect the structure of stem 1b with minor disruption of immediately adjacent structures (IL1 and IL2)+ The weak but detectable binding of PSF and p54 nrb to the 39 mutant implies that regions other than stem 1b might also be involved in binding+ Association of PSF and p54 nrb with U5 snRNA during splicing Double-stranded RNAs adopt the A-form conformation that precludes base-specific interaction with protein side chains in the deep major groove (Steitz, 1999)+ Therefore, the question arises as to how PSF and p54 nrb interact with an A-form helix while apparently maintaining sequence specificity+ One speculative possibility is that PSF and p54 nrb could bind U5 in two ways: to the intact stem, or to the 39 side of the stem after melting+ At least two ATPases (U5-100 kDa and U5-200 kDa) and one homolog of EF-2 GTPase (U5-116 kDa) have been found to associate with U5 snRNP and may function to mediate unwinding to facilitate the multiple RNA-RNA rearrangements that are central to splicing (Fabrizio et al+, 1997;Teigelkamp et al+, 1997;Laggerbauer et al+, 1998)+ Interestingly, hPrp8 (U5-220 kDa), which makes multiple contacts with the pre-mRNA and with U5 (MacMillan et al+, 1994;Reyes et al+, 1996;Chiara et al+, 1997), forms a stable complex with three U5 proteins (U5-200, U5-116, and U5-40; Achsel et al+, 1998)+ IL2, the internal loop between stems 1b and 1c, is required for efficient association of hPrp8 and U5-116 kDa with U5 (Hinz et al+, 1996;Ségault et al+, 1999)+ Given that the U5-200 kDa protein is a putative unwindase, it seems reasonable to propose that stem 1b of U5 might also undergo a conformational change during spliceosome assembly or during the two catalytic steps+ Interestingly, two-hybrid screens using fragments of hPrp8 have detected interaction with PSF (G+ Moreau and M+ Moore, pers+ comm+), consistent with association of these two proteins to U5 in the vicinity of stem 1b+…”

“…The high degree of conservation of U5 stem 1b in vertebrates and flies implies that it plays a critical role in U5 function+ Ségault et al+ (1999) examined the ability of several human U5 snRNA mutants to function in splicing by reconstituting U5-depleted nuclear extract with in vitro transcribed mutant U5 snRNAs+ Although the rescue of splicing was prevented by deletion of IL2, a stem 1b mutant (sub-stem 1b) was still partially functional, perhaps suggesting that this region is not important after all+ However, the sub-stem 1b mutant maintained a purine-rich sequence of 59-CAGAGA GAAGU-39 on the 59 side of the stem+ Comparison of this sequence with the 39 strand of the original stem (the optimal PSF-p54 nrb binding site) showed that all the changes are transitions, whereas most of our changes are transversions+ Furthermore, about 50% of the SELEX sequences we identified contain at least one AGAG or GAAG motifs (Fig+ 2A,B,C)+ Thus, it is possible that the sub-stem 1b mutant fortuitously maintained a binding site for PSF and p54 nrb on the 59 side of stem 1b+…”

The carboxy terminal WD domain of the pre-mRNA splicing factor Prp17p is critical for function

“…In yeast, the second-step factors have been ordered with respect to an ATP-dependent event during the second step (Schwer & Guthrie, 1991;Horowitz & Abelson, 1993;Ansari & Schwer, 1995;Jones et al+, 1995)+ The RNA helicase Prp16p hydrolyzes ATP and is required for a conformational change that occurs in the spliceosome after the first step (Schwer & Guthrie, 1992)+ yPrp17p is required before or during this event; Prp18p and Slu7p are required afterwards (Umen & Guthrie, 1995b)+ A role for the yeast Prp22 in the second step of splicing has been shown more recently (Schwer & Gross, 1998), and it is proposed to act in conjunction with Slu7 and Prp18+ Rearrangements in the spliceosome after the first step are required to identify and position the 39 splice site for the second step+ After the first step of splicing, the highly conserved loop I of U5 snRNA plays an important role in aligning the two exons for the second-step catalytic site+ In yeast, this loop is essential for the second step; however, in humans it is not required (O'Keefe et al+, 1996;Segault et al+, 1999)+ The loop I uridines are likely involved in noncanonical base pairing with the exon sequences near the splice sites, which are weakly conserved compared to elements in the intron+ Additional interactions appear to be necessary for stabilization of the U5 snRNA and the two exons during the second step+ For example, the highly conserved U5 snRNP protein Prp8p crosslinks to both the 59 and 39 splice sites (Wyatt et al+, 1992;Teigelkamp et al+, 1995;Umen & Guthrie, 1995a, 1995b)+ In addition, Slu7p and yPrp17p have been linked genetically to the function of U5 snRNA loop I Seshadri et al+, 1996)+ Prp18p and Slu7p have been shown to physically interact (Zhang & Schwer, 1997); however, no other direct interactions have been shown between proteins required for the second step+ Studies of synthetic lethality suggest that there is a functional interaction between all the second-step factors (reviewed in Umen & Guthrie, 1995c)+ Alleles of PRP17 are synthetically lethal with alleles of SLU7, PRP16, PRP18, PRP8, SLT11, U5 snRNA, and U2 snRNA Umen & Guthrie, 1995a;Seshadri et al+, 1996;Xu et al+, 1998), implying specific, and possibly direct interactions+ Insufficient knowledge of the proteins and/or RNAs that interact with yPrp17p, and the function of this protein, severely limits our understanding of its role in the second step of pre-mRNA splicing+ Through co...…”

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

“…In Saccharomyces cerevisiae, the 5Ј exon-U5 snRNA interaction is thought important for both retention of the 5Ј exon intermediate and alignment of the 5Ј and 3Ј exons prior to exon ligation (Newman and Norman 1992). In mammalian extracts, however, U5 snRNA loop I can be deleted entirely with no ill effect on either step of splicing (O'Keefe et al 1996;Segault et al 1999). Thus, the mammalian spliceosome must use interactions in addition to any base pairings with U5 snRNA to maintain its grip on the 5Ј exon intermediate and properly align the second-step reactants.…”