To ligate exons in pre-messenger RNA (pre-mRNA) splicing, the spliceosome must reposition the substrate after cleaving the 5 splice site. Because spliceosomal small nuclear RNAs (snRNAs) bind the substrate, snRNA structures may rearrange to reposition the substrate. However, such rearrangements have remained undefined. Although U2 stem IIc inhibits binding of U2 snRNP to pre-mRNA during assembly, we found that weakening U2 stem IIc suppressed a mutation in prp16, a DExD/H box ATPase that promotes splicing after 5 splice site cleavage. The prp16 mutation was also suppressed by mutations flanking stem IIc, suggesting that Prp16p facilitates a switch from stem IIc to the mutually exclusive U2 stem IIa, which activates binding of U2 to pre-mRNA during assembly. Providing evidence that stem IIa switches back to stem IIc before exon ligation, disrupting stem IIa suppressed 3 splice site mutations, and disrupting stem IIc impaired exon ligation. Disrupting stem IIc also exacerbated the 5 splice site cleavage defects of certain substrate mutations, suggesting a parallel role for stem IIc at both catalytic stages. We propose that U2, much like the ribosome, toggles between two conformations-a closed stem IIc conformation that promotes catalysis and an open stem IIa conformation that promotes substrate binding and release.[Keywords: U2; snRNA; Prp16; spliceosome; DExD/H box; pre-mRNA splicing]Received January 30, 2007; revised version accepted February 18, 2007. Introns are excised from pre-messenger RNA (premRNA) by the spliceosome, a large ribonucleoprotein machine composed of >100 proteins and five small nuclear RNAs (snRNAs) (for reviews, see Jurica and Moore 2003;Will and Lührmann 2006). The spliceosome excises introns in two sequential transesterification reactions. In the first chemical step, termed 5Ј splice site cleavage, the 2Ј hydroxyl of an intronic adenosine, termed the branch point, attacks the 5Ј splice site, forming a lariat intermediate and a liberated 5Ј exon with a free 3Ј hydroxyl. In the second chemical step, termed exon ligation, the 3Ј hydroxyl of the 5Ј exon attacks the 3Ј splice site, excising the lariat intron and ligating the exons to form mRNA. Because the leaving group of the first reaction becomes the attacking group for the second reaction, this two-step reaction presents a significant biochemical challenge to the spliceosome, which must consequently rearrange the substrate after 5Ј splice site cleavage (for review, see Staley and Guthrie 1998). Specifically, because the spliceosome is thought to catalyze the two similar reactions in one active site, and the second reaction is essentially the reverse of the first (Steitz and Steitz 1993), the spliceosome likely removes the branched product of 5Ј splice site cleavage from the active site and replaces the branch with the 3Ј splice site. The mechanism by which the spliceosome rearranges the substrate is understood poorly.The substrate is defined by intronic consensus sequences at the 5Ј splice site, the branch site, and the 3Ј splice site (f...

To catalyze pre-mRNA splicing, U6 snRNA positions two metals that interact directly with the scissile phosphates. The U6 metal ligands correspond stereospecifically to metal ligands within the catalytic domain V of a group II self-splicing intron. In domain V, the ligands are organized by base-triple interactions, which also juxtapose the 3′ splice site with the catalytic metals. However, in the spliceosome, the mechanism for organizing catalytic metals and recruiting the substrate has remained unclear. Here we show by genetics, crosslinking, and biochemistry in yeast that analogous triples form in U6 and promote catalytic metal binding and both chemical steps of splicing. Because the triples include an element that defines the 5′ splice site, the triples also provide a mechanism for juxtaposing the pre-mRNA substrate with the catalytic metals. Our data indicate that U6 adopts a group II intron-like tertiary conformation to catalyze splicing.

During pre-mRNA splicing, the spliceosome must configure the substrate, catalyze 59 splice site cleavage, reposition the substrate, and catalyze exon ligation. The highly conserved U2/U6 helix I, which adjoins sequences that define the reactive sites, has been proposed to configure the substrate for 59 splice site cleavage and promote catalysis. However, a role for this helix at either catalytic step has not been tested rigorously and previous observations question its role at the catalytic steps. Through a comprehensive molecular genetic study of U2/U6 helix I, we found that weakening U2/U6 helix I, but not mutually exclusive structures, compromised splicing of a substrate limited at the catalytic step of 59 splice site cleavage, providing the first compelling evidence that this helix indeed configures the substrate during 59 splice site cleavage. Further, mutations that we proved weaken only U2/U6 helix I suppressed a mutation in PRP16, a DEAH-box ATPase required after 59 splice site cleavage, providing persuasive evidence that helix I is destabilized by Prp16p and suggesting that this structure is unwound between the catalytic steps. Lastly, weakening U2/U6 helix I also compromised splicing of a substrate limited at the catalytic step of exon ligation, providing evidence that U2/U6 helix I reforms and functions during exon ligation. Thus, our data provide evidence for a fundamental and apparently dynamic role for U2/U6 helix I during the catalytic stages of splicing.

Telomerase is a specialized chromosome end-replicating enzyme required for genome duplication in many eukaryotes. An RNA and reverse transcriptase protein subunit comprise its enzymatic core. Telomerase is evolving rapidly, particularly its RNA component. Nevertheless, nearly all telomerase RNAs, including those of H. sapiens and S. cerevisiae, share four conserved structural elements: a core-enclosing helix (CEH), template-boundary element, template, and pseudoknot, in this order along the RNA. It is not clear how these elements coordinate telomerase activity. We find that although rearranging the order of the four conserved elements in the yeast telomerase RNA subunit, TLC1, disrupts activity, the RNA ends can be moved between the template and pseudoknot in vitro and in vivo. However, the ends disrupt activity when inserted between the other structured elements, defining an Area of Required Connectivity (ARC). Within the ARC, we find that only the junction nucleotides between the pseudoknot and CEH are essential. Integrating all of our findings provides a basic map of functional connections in the core of the yeast telomerase RNP and a framework to understand conserved element coordination in telomerase mechanism.