Primary transcripts made by RNA polymerase II (Pol II), but not Pol I or Pol III, are modified by addition of a 7-methylguanosine (m7G) residue to the triphosphate 5′ end shortly after it emerges from the polymerase. The m7G “caps” of small nuclear and small nucleolar RNAs, but not messenger RNAs, are subsequently hypermethylated to a 2,2,7-trimethylguanosine (TMG) residue. U6 RNA, the only small nuclear RNA synthesized by Pol III in most eukaryotes, does not receive a methylguanosine cap. However, human U6 RNA is O-methylated on the 5′-terminal (γ) phosphate by an enzyme that recognizes the 5′ stem-loop of U6. Here we show that variant yeast U6 RNAs truncated or substituted within the 5′ stem-loop are TMG capped in vivo. Accumulation of the most efficiently TMG-capped U6 RNA variant is strongly inhibited by a conditional mutation in the largest subunit of Pol III, confirming that it is indeed synthesized by Pol III. Thus, methylguanosine capping and cap hypermethylation are not exclusive to Pol II transcripts in yeast. We propose that TMG capping of variant U6 RNAs occurs posttranscriptionally due to exposure of the 5′ triphosphate by disruption of protein binding and/or γ-methyl phosphate capping. 5′ truncation and TMG capping of U6 RNA does not appear to affect its normal function in splicing, suggesting that assembly and action of the spliceosome is not very sensitive to the 5′ end structure of U6 RNA.
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