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Molecular and Cellular Biology, December 1998, p. 6885-6896, Vol. 18, No. 12
Laboratories of Genetics and Molecular
Biology, University of Wisconsin, Madison, Wisconsin 53706
Received 22 June 1998/Returned for modification 17 August
1998/Accepted 15 September 1998
Sen1p from Saccharomyces cerevisiae is a nucleic acid
helicase related to DEAD box RNA helicases and type I DNA helicases. The temperature-sensitive sen1-1 mutation located in the
helicase motif alters the accumulation of pre-tRNAs, pre-rRNAs, and
some small nuclear RNAs. In this report, we show that cells carrying sen1-1 exhibit altered accumulation of several small
nucleolar RNAs (snoRNAs) immediately upon temperature shift. Using
Northern blotting, RNase H cleavage, primer extension, and base
compositional analysis, we detected three forms of the snoRNA snR13 in
wild-type cells: an abundant TMG-capped 124-nucleotide (nt) mature form (snR13F) and two less abundant RNAs, including a heterogeneous population of ~1,400-nt 3'-extended forms (snR13R) and a 108-nt 5'-truncated form (snR13T) that is missing 16 nt at the 5' end. A
subpopulation of snR13R contains the same 5' truncation. Newly synthesized snR13R RNA accumulates with time at the expense of snR13F
following temperature shift of sen1-1 cells, suggesting a
possible precursor-product relationship. snR13R and snR13T both increase in abundance at the restrictive temperature, indicating that
Sen1p stabilizes the 5' end and promotes maturation of the 3' end.
snR13F contains canonical C and D boxes common to many snoRNAs. The 5'
end of snR13T and the 3' end of snR13F reside within
C2U4 sequences that immediately flank the C and
D boxes. A mutation in the 5' C2U4 repeat
causes underaccumulation of snR13F, whereas mutations in the 3'
C2U4 repeat cause the accumulation of two novel
RNAs that migrate in the 500-nt range. At the restrictive temperature,
double mutants carrying sen1-1 and mutations in the 3'
C2U4 repeat show reduced accumulation of the
novel RNAs and increased accumulation of snR13R RNA, indicating that
Sen1p and the 3' C2U4 sequence act in a common
pathway to facilitate 3' end formation. Based on these findings, we
propose that Sen1p and the C2U4 repeats that
flank the C and D boxes promote maturation of the 3' terminus and
stability of the 5' terminus and are required for maximal rates of
synthesis and levels of accumulation of mature snR13F.
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
The Putative Nucleic Acid Helicase Sen1p Is Required for
Formation and Stability of Termini and for Maximal Rates of Synthesis
and Levels of Accumulation of Small Nucleolar RNAs in
Saccharomyces cerevisiae
and
*
Corresponding author. Mailing address: R. M. Bock
Laboratories, 1525 Linden Dr., University of Wisconsin, Madison, WI
53706. Phone: (608) 262-5388. Fax: (608) 262-4570. E-mail:
mrculber{at}facstaff.wisc.edu.
University of Wisconsin Laboratory of Genetics paper 3486.
Present address: Whitehead Institute for Biomedical Research, Nine
Cambridge Center, Cambridge, MA 02142.
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