Disruption of the 5 ' stem-loop of yeast U6 RNA induces trimethylguanosinecapping of this RNA polymerase III transcript in vivo

Primary transcripts made by RNA polymerase II (Pol II), but not Pol I or Po l III, are modified by addition of a 7-methylguanosine (m(7)G) residue to t he triphosphate 5' end shortly after it emerges from the polymerase. The m( 7)G "caps" of small nuclear and small nucleolar RNAs, but not messenger RNA s, ave subsequently hypermethylated to a 2,2,7-trimethylguanosine (TMG) res idue. U6 RNA, the only small nuclear RNA synthesized by pol III in most euk aryotes, does not receive a methylguanosine cap. However, human U6 RNA is O -methylated on the 5'-terminal (gamma) phosphate by an enzyme that recogniz es the 5' stem-loop of U6. Here we show that variant yeast U6 RNAs truncate d or substituted within the 5' stem-loop are TMG capped in vivo. Accumulati on of the most efficiently TMG-capped U6 RNA variant is strongly inhibited by a conditional mutation in the largest subunit of Pol III, confirming tha t it is indeed synthesized by Pol III. Thus, methylguanosine capping and ca p hypermethylation are not exclusive to Pol II transcripts in yeast. We pro pose that TMG capping of variant U6 RNAs occurs posttranscriptionally due t o exposure of the 5' triphosphate by disruption of protein binding and/or g amma -methyl phosphate capping. 5' truncation and TMG capping of U6 RNA doe s not appear to affect its normal function in splicing, suggesting that ass embly and action of the spliceosome is not very sensitive to the 5' end str ucture of U6 RNA.