SPLICEOSOMAL snRNAs

Transcription on lampbrush chromosome loops in the absence of U2 snRNA.

“…Data from in vitro splicing experiments stress the sequential addition of splicing components to the premRNA (Maniatis and Reed, 1987;Guthrie and Patterson, 1988;Steitz et al, 1988;Ruby and Abelson, 1991). The U1 snRNP first binds to the 5' splice junction; the U2 snRNP then associates with the lariat region; and finally, the U5 and U4/U6 snRNPs are added, building up the fully formed spliceosome.…”

Conservation of functional features of U6atac and U12 snRNAs between vertebrates and higher plants

“…To determine if some of the differences noted above between plant and human U6atac snRNAs were functionally silent, we tested them in the context of an in vivo suppression assay for U6atac snRNA+ We have previously shown that human U6atac snRNA compensatory mutants expressed in Chinese hamster ovary (CHO) cells can suppress the in vivo cryptic splicing phenotype of 59 splice site mutants of a U12-dependent intron (Incorvaia & Padgett, 1998)+ Starting with this suppressor snRNA, we tested the effect of the A-to-G difference at position 26 of plant U6atac+ This position appears to be homologous to the invariant A of the AGC motif found in U6 snRNAs (Brow & Guthrie, 1988) and in human U6atac snRNAs (Tarn & Steitz, 1996b)+ In yeast U6 snRNA, mutation of this position leads to defects in splicing, particularly in the second step (Fabrizio & Abelson, 1990;Madhani et al+, 1990)+ However, mutation of this residue in mammalian U6 snRNA had no effect using an in vivo suppression assay (Datta & Weiner, 1993)+ When the A 26-to-G mutation was introduced into the suppressor U6atac construct (Figs+ 8A and 9), full in vivo suppressor activity was maintained showing that G 26 is fully compatible with active U12-dependent splicing+ Note that both A 26 and G 26 can potentially base pair to U12 snRNA in slightly different registers (Figs+ 7 and 8A)+ Immediately following this (A/G)GC sequence is a region that can form an intramolecular stem-loop that is similar in size, position, and structure to a critical region of U6 snRNA+ Although the plant and human U6atac sequences differ by almost 50% in this region, the predicted structures are similar+ To demonstrate that the plant stem-loop could still be active in spite of these differences, we constructed a chimeric U6atac in which the plant stem-loop replaced the human stemloop+ The resulting construct was tested for activity in vivo using the same suppressor assay described above+ We found that, in the presence of a mutated human U4atac snRNA, this chimeric U6atac snRNA was active in vivo (Figs+ 8A and 9)+ This shows that the function of the plant intramolecular stem-loop structure is conserved+ These data also provide the first in vivo evidence for the predicted function of U4atac snRNA in U12-dependent splicing+ When Tarn and Steitz (1996b) identified U6atac and U4atac snRNAs in human nuclear extracts, they noted that the two snRNAs could adopt a base-paired structure analogous to that formed by U4 and U6 snRNAs+ In the case of U4/U6 snRNA, this structure appears to be required for splicing in vivo and in vitro (Wolff & Bindereif, 1992)+ The precise role of this structure is still unclear but it has been proposed that U4 snRNA acts as a chaperone to deliver the U6 snRNA to the nascent spliceosome in an inactive form (Guthrie & Patterson, 1988)+ Subsequently, through the action of ATP-dependent helicases (Raghunathan & Guthrie, 1998), the two snRNAs are separated and U6 goes on to form alternative base-pairing interactions with U2 and the intron 59 splice site whereas U4 appears to be destabilized from the spliceosome (Lamond et al+, 1988;Yean &...…”

“…The presence of multiple introns in most nuclear mRNA coding genes is a distinctive feature of the genomes of animals and higher plants+ The consensus features of splice sites in these two groups of organisms are very similar, although there may be some differences in the mechanism of splice site definition (Wiebauer et al+, 1988;Simpson & Filipowicz, 1996;Brown & Simpson, 1998)+ Even more striking is the conservation of the sequences and structures of the small nuclear RNAs that are involved in spliceosome structure and function (Brow & Guthrie, 1988;Guthrie & Patterson, 1988;Reddy & Busch, 1988)+ The most conserved regions of these snRNAs are the portions of U6 and U2 that are believed to comprise the central set of RNA-RNA interactions in the spliceosome (Nilsen, 1998)+ In these regions, the sequences of human and plant snRNAs are almost identical+ Although this conservation of sequences over the billion years of evolution that separate these taxonomic groups testifies to their important function in splicing, the lack of variation makes it difficult to use phylogenetic covariation to substantiate potential RNA-RNA interactions+…”

A quantitative study of the flexibility contributed to RNA structures by nicks and single-stranded gaps

“…The cleavage site of the hammerhead ribozyme occurs at a two-helix junction (Forster & Symons, 1987)+ Cleavage of the RNA substrate is favored over ligation of the products, suggesting that cleavage increases the extent of thermal motion in the hammerheadoligonucleotide complex, thereby making ligation entropically disfavored (Hertel et al+, 1994)+ Our results qualitatively support this interpretation, although tertiary interactions not present in the model duplexes are expected to modulate the frequency, extent, and direction of thermal motions in more complex RNA molecules+ Two-helix junctions also occur in the reactants for ligation in the hairpin ribozyme (Hampel et al+, 1990) and within P1 of group I introns after the first step of splicing (Cech et al+, 1994)+ Two helices separated by one or more single-stranded nucleotides are found in many other natural RNAs, including RNase P (James et al+, 1988) and the snRNAs U2 (Guthrie & Patterson, 1988) and U8 (Sollner-Webb et al+, 1993)+ Some of the aforementioned RNAs exist as ribonucleoprotein complexes and may display dynamic behavior different from that of the free RNA+ Comparing the conformational dynamics of free RNAs with their corresponding RNPs represents another application of the thiol-disulfide interchange chemistry to be pursued in future studies+…”