A dynamic bulge in the U6 RNA internal stem–loop functions in spliceosome assembly and activation

Abstract

The highly conserved internal stem-loop (ISL) of U6 spliceosomal RNA is unwound for U4/U6 complex formation during spliceosome assembly and reformed upon U4 release during spliceosome activation. The U6 ISL is structurally similar to Domain 5 of group II self-splicing introns, and contains a dynamic bulge that coordinates a Mg ++ ion essential for the first catalytic step of splicing. We have analyzed the causes of growth defects resulting from mutations in the Saccharomyces cerevisiae U6 ISL-bulged nucleotide U80 and the adjacent C67-A79 base pair. Intragenic suppressors and enhancers of the cold-sensitive A79G mutation, which replaces the C-A pair with a C-G pair, suggest that it stabilizes the ISL, inhibits U4/U6 assembly, and may also disrupt spliceosome activation. The lethality of mutations C67A and C67G results from disruption of base-pairing potential between U4 and U6, as these mutations are fully suppressed by compensatory mutations in U4 RNA. Strikingly, suppressor analysis shows that the lethality of the U80G mutation is due not only to formation of a stable base pair with C67, as previously proposed, but also another defect. A U6-U80G strain in which mispairing with position 67 is prevented grows poorly and assembles aberrant spliceosomes that retain U1 snRNP and fail to fully unwind the U4/U6 complex at elevated temperatures. Our data suggest that the U6 ISL bulge is important for coupling U1 snRNP release with U4/U6 unwinding during spliceosome activation.

Spliceosome assembly in the absence of stable U4/U6 RNA pairing

“…3;Li and Brow 1993). As shown previously (Fortner et al 1994;McManus et al 2007), in wild-type cells 70%-85% of U4 RNA is paired with U6 RNA (lanes 1,2). Both U6-A62G (lanes 3,4) and U4-G14U (lanes 9,10) decrease the fraction of U4 RNA base-paired to U6 RNA even when cells are grown at permissive temperature (30°C), although only U6-A62G has a statistically significant effect in this study.…”

“…2B, row 4), particularly when the U4 gene is on a plasmid and can therefore be amplified (Fortner et al 1994), as is the case with the strain used here. Because both U4-G14U and U6-A62G result in a decrease in U4/U6 complex formation (Fortner et al 1994;McManus et al 2007), we hypothesized that U6-A91G might also suppress U6-A62G. An extensive but nonsaturating selection for suppressors of the cold-sensitivity of U6-A62G yielded mutations at 15 different positions in U6 RNA, but none in A91 (Fortner et al 1994).…”

“…The fact that both U4-G14U and U6-A62G inhibit U4/U6 di-snRNP assembly and/or decrease U4/U6 stability in vivo (Shannon and Guthrie 1991;Vidaver et al 1999;McManus et al 2007) suggested that, in combination, the two mutations would have a stronger growth defect than either alone. Indeed, the U4-G14U/U6-A62G mutation is dead at 30°C (Fig.…”

“…pRS314-U4+U6 was created by transferring the SpeI/EcoRI fragment of pRS316-U4wt-U6mini (McManus et al 2007) to pRS314. pRS313-U4, pRS313-U4-G14U, pRS314-U6, and pRS314-U6-A62G have been described previously (Fortner et al 1994;Kuhn and Brow 2000).…”

“…Mutant U4 and U6 alleles were introduced into Saccharomyces cerevisiae strain CJM000 (Gietz et al 1995;McManus et al 2007) individually in plasmids pRS313 and pRS314, respectively, or together in pRS314-U4+U6. Transformants were streaked onto SC medium containing 0.75 mg/mL 5-fluoroorotic acid (5-FOA) to select for strains containing only the newly acquired alleles.…”