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

Abstract

Splicing of U12-dependent introns requires the function of U11, U12, U6atac, U4atac, and U5 snRNAs. Recent studies have suggested that U6atac and U12 snRNAs interact extensively with each other, as well as with the pre-mRNA by Watson-Crick base pairing. The overall structure and many of the sequences are very similar to the highly conserved analogous regions of U6 and U2 snRNAs. We have identified the homologs of U6atac and U12 snRNAs in the plant Arabidopsis thaliana. These snRNAs are significantly diverged from human, showing overall identities of 65% for U6atac and 55% for U12 snRNA. However, there is almost complete conservation of the sequences and structures that are implicated in splicing. The sequence of plant U6atac snRNA shows complete conservation of the nucleotides that base pair to the 59 splice site sequences of U12-dependent introns in human. The immediately adjacent AGAGA sequence, which is found in human U6atac and all U6 snRNAs, is also conserved. High conservation is also observed in the sequences of U6atac and U12 that are believed to base pair with each other. The intramolecular U6atac stem-loop structure immediately adjacent to the U12 interaction region differs from the human sequence in 9 out of 21 positions. Most of these differences are in base pairing regions with compensatory changes occurring across the stem. To show that this stem-loop was functional, it was transplanted into a human suppressor U6atac snRNA expression construct. This chimeric snRNA was inactive in vivo but could be rescued by coexpression of a U4atac snRNA expression construct containing compensatory mutations that restored base pairing to the chimeric U6atac snRNA. These data show that base pairing of U4atac snRNA to U6atac snRNA has a required role in vivo and that the plant U6atac intramolecular stem-loop is the functional analog of the human sequence.

The intramolecular stem-loop structure of U6 snRNA can functionally replace the U6atac snRNA stem-loop

“…We have previously developed an in vivo mutational suppressor assay for the function of several of the snRNAs involved in U12-dependent splicing (Hall & Padgett, 1996;Kolossova & Padgett, 1997;Incorvaia & Padgett, 1998;Shukla & Padgett, 1999)+ This assay relies on the genetic suppression of splicing defects due to splice site mutations in a U12-dependent intron by coexpression of compensatory mutant snRNAs+ For U6atac snRNA, a mutation in the 59 splice site of a transfected minigene construct, which blocks splicing at the normal sites, is suppressed by cotransfection of expression constructs containing compensatory mutations in U11 and U6atac snRNAs+ The elements of this assay are diagramed in Figure 1B+ The P120 minigene contains a double mutation in positions 5 and 6 of the 59 splice site of the U12-dependent intron F (P120 CC5/ 6GG)+ This mutation causes a complete loss of splicing activity at the normal 59 and 39 splice sites and the concomitant activation of a pair of cryptic splice sites within the intron (Kolossova & Padgett, 1997)+ In vitro FIGURE 2. Comparison of the intramolecular stem-loop structures of various U6 and U6atac snRNAs+ The sequences are from human, A. thaliana (plant), and Saccharomyces cerevisiae (yeast)+ In each case the putative helix Ib interactions with the conspecific U2 or U12 snRNAs are shown below the intramolecular structures+ The boxed sequences are those that were substituted into the human U6atac snRNA in our previous analysis of the plant U6atac stem-loop (Shukla & Padgett, 1999) or in the work discussed here+ analysis has shown that this cryptic splicing reaction is catalyzed by the U2-dependent spliceosome (Tarn & Steitz, 1996a)+ Cotransfection of U6atac and U11 snRNA expression constructs that contain the compensatory mutations shown in Figure 1B restores U12-dependent splicing at the normal 59 and 39 splice sites to nearly wild-type levels+ This suppression is completely dependent on the U6atac suppressor while the U11 suppressor improves the level of suppression (Incorvaia & Padgett, 1998)+ This dependence on the addition of an exogenous suppressor U6atac allows us to assay the in vivo effects of mutations at other sites in U6atac snRNA+…”

“…Comparison of the intramolecular stem-loop structures of various U6 and U6atac snRNAs+ The sequences are from human, A. thaliana (plant), and Saccharomyces cerevisiae (yeast)+ In each case the putative helix Ib interactions with the conspecific U2 or U12 snRNAs are shown below the intramolecular structures+ The boxed sequences are those that were substituted into the human U6atac snRNA in our previous analysis of the plant U6atac stem-loop (Shukla & Padgett, 1999) or in the work discussed here+ analysis has shown that this cryptic splicing reaction is catalyzed by the U2-dependent spliceosome (Tarn & Steitz, 1996a)+ Cotransfection of U6atac and U11 snRNA expression constructs that contain the compensatory mutations shown in Figure 1B restores U12-dependent splicing at the normal 59 and 39 splice sites to nearly wild-type levels+ This suppression is completely dependent on the U6atac suppressor while the U11 suppressor improves the level of suppression (Incorvaia & Padgett, 1998)+ This dependence on the addition of an exogenous suppressor U6atac allows us to assay the in vivo effects of mutations at other sites in U6atac snRNA+…”

“…The precise mutations that were made for the U6/ U6atac chimeras are shown in Figure 3A+ In our earlier test of chimeric human/plant U6atac snRNAs, we found that the chimeras were inactive in vivo unless compensating alterations were made to the human U4atac snRNA sequence and this snRNA was coexpressed in vivo with the chimeric U6atac snRNA (Shukla & Padgett, 1999)+ Thus, for each of the U6/ U6atac chimeras, we constructed analogous modified U4atac snRNA expression constructs as shown in Figure 3A+ In designing these U4atac constructs, we attempted to maintain the amount of pairing and a similar overall structure to that predicted to form between the native U4atac and U6atac snRNAs+…”

“…The first clues to the functional significance of this region of U6atac came from a comparison of the human and plant U6atac snRNAs (Shukla & Padgett, 1999)+ This comparison showed that, although the sequences in this region differed by almost 50% between the two organisms, the overall structure was maintained by compensatory mutations in the putative basepaired stem regions whereas the loop and bulge nucleotides were largely conserved+ To show that the plant element retained the same function as the human element, a chimeric U6atac snRNA was constructed with the plant stem-loop sequence in the background of the human U6atac snRNA+ By adding a suppressor mutation that allowed us to assess the function of this chimeric snRNA in vivo, we showed that the plant stemloop sequence functioned in the human snRNA (Shukla & Padgett, 1999)+ In the work described here, we show that the human U6atac snRNA stem-loop structure is important for in vivo splicing+ Several mutations in the suppressor U6atac snRNA abolish in vivo function+ These include mutations that are expected to lead to mispairing within the stems and mutations that remove one or both of the bulged nucleotides+ The splicing defects of the mispairing mutations could be reversed by restoring the potential for base pairing with compensatory mutations FIGURE 6. In vivo functional analysis of mutant U6atac snRNA constructs that delete or change the bases in the bulge region+ A: Location of the mutations in the U6atac intramolecular stem-loop structure for the bulge deletions and mutation+ B: Diagram showing the mutations made in human U6atac and U4atac snRNAs for the bulge deletions and mutation+ Each mutation was constructed alone and in combination+ Not shown is the U6atac GG14/15CC suppressor mutation that was also included in the constructs+ C: In vivo splicing assay of the modified snRNAs+ Refer to Figure 3B for details+ The indicated mutant U6atac snRNAs were cotransfected in lanes 8-17 along with the U11 GG6/ 7CC suppressor without (lanes 8, 10, 12, and 14) or with (lanes 9, 11, 13, and 15) the corresponding U4atac snRNA suppressor constructs+ on the other side of the stems+ These results confirm the prediction of the phylogenetic comparisons between the human and plant sequences+…”

“…Comparison of RNA-RNA interactions in U2-and U12-dependent spliceosomal splicing+ A: Diagram of the interactions between the pre-mRNA and U1, U2, and U6 snRNAs+ Shown are the U2-branch site interaction, the U1 and U6-59 splice site interactions and the Helix Ia and Ib interactions between U2 and U6 snRNAs+ Also shown is the U6 intramolecular stem-loop structure that immediately follows Helix Ib+ B: Diagram of the analogous interactions between the pre-mRNA and U11, U12, and U6atac snRNAs+ Shown are the U12-branch site interaction, the U11 and U6atac-59 splice site interactions and the U12-U6atac interactions and the U6atac stem-loop structure+ Also shown in the shaded boxes are the mutations used in the in vivo mutational suppression assay+ The P120 CC5/6GG mutation shown in the middle of the upper box inactivates U12-dependent splicing+ The U6atac GG14/15CC mutation restores splicing at the mutant 59 splice site and the U11 GG6/7CC mutation enhances the level of suppression+ functionally active snRNA when tested in vivo (Shukla & Padgett, 1999)+ In addition to the indirect evidence of the importance of the U6 snRNA intramolecular stem-loop provided by phylogenetic conservation, experimental support has also been provided by several studies+ Genetic suppression experiments in yeast have shown that formation of the stem-loop structure is required for splicing (Fortner et al+, 1994;McPheeters, 1996)+ Similar experiments in mammalian systems also showed a requirement for this structure (Wolff & Bindereif, 1993;Sun & Manley, 1995+ In an extensive set of experiments, Sun and Manley (1997) used an in vivo approach to show that U6 snRNA function was maintained as long as the base pairing pattern and a critical U residue in the bulge were conserved+ Other structural modifications were also compatible with function including pairing of the bulged U residue and extension of the helix by an additional base pair+ These results are quite surprising in light of the very high conservation of this region over more than a billion years of evolution+ Results from in vitro modification studies of residues within the U6 intramolecular stem-loop provide additional support for its role in splicing+ In both yeast (Fabrizio & Abelson, 1992) and nematode (Yu et al+, 1995) in vitro splicing systems, phosphorothioate modification of certain phosphodiester bonds blocks splicing+ Such a block could be due to disruption of either RNAprotein interactions or interactions with functional chemical groups such as metal ions required for catalysis or the maintenance of a catalytically active structure (Eckstein, 1985)+ A recent analysis of one of these positions in yeast U6 snRNA has identified a critical metal-ion binding site in the bulge region (Yean et al+, 2000)+ These and other results have led to speculation that this element of U6 snRNA functions at or near the catalytic center of the spliceosome (reviewed in Nilsen, 1998;Collins & Guthrie, 2000)+ Because our earlier experience with substituting the plant U6atac snRNA stem-loop into human U6atac suggested that there was significant sequence flexibility ...…”