Characterization of the essential activities of Saccharomyces cerevisiae Mtr4p, a 3'->5' helicase partner of the nuclear exosome.
Mtr4p belongs to the Ski2p family of DEVH-box containing proteins and is required for processing and degradation of a variety of RNA substrates in the nucleus. In particular, Mtr4p is required for creating the 5.8 S ribosomal RNA from its 7 S precursor, proper 3'-end processing of the U4 small nuclear RNA and some small nucleolar RNAs, and degradation of aberrant mRNAs and tRNAs. In these studies we have shown that Mtr4p has RNA-dependent ATPase (or dATPase) activity that is stimulated effectively by likely substrates (e.g. tRNA) but surprisingly weakly by poly(A). Using an RNA strand-displacement assay, we have demonstrated that Mtr4p can, in the presence of ATP or dATP, unwind the duplex region of a partial duplex RNA substrate in the 3'-->5' direction. We have examined the ability of Mtr4p to bind model RNA substrates in the presence of nucleotides that mimic the stages (i.e. ATP-bound, ADP-bound, and nucleotide-free) of the unwinding reaction. Our results demonstrate that the presence of a non-hydrolyzable ATP analog allows Mtr4p to discriminate between partial duplex RNA substrates, binding a 3'-tailed substrate with 5-fold higher affinity than a 5'-tailed substrate. In addition, Mtr4p displays a marked preference for binding to poly(A) RNA relative to an oligoribonucleotide of the same length and a random sequence. This binding exhibits apparent cooperativity and different dynamic behavior from binding to the random single-stranded RNA. This unique binding mode might be employed primarily for degradation.
Bernstein
Jade
J
Department of Biochemistry and Molecular Biology, and Marlene and Stewart Greenebaum Cancer Center, University of Maryland School of Medicine, Baltimore, Maryland 21201, USA.
Patterson
Dimeka N
DN
Wilson
Gerald M
GM
Toth
Eric A
EA
D000244:Adenosine Diphosphate / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000235:genetics / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D002467:Cell Nucleus / Q000235:genetics / Q000378:metabolism*; D053487:DEAD-box RNA Helicases / Q000235:genetics / Q000378:metabolism*; D012323:RNA Processing, Post-Transcriptional / Q000502:physiology*; D012331:RNA, Fungal / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity / Q000502:physiology
D012331:RNA, Fungal; D029701:Saccharomyces cerevisiae Proteins; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; C101004:MTR4 protein, S cerevisiae; D053487:DEAD-box RNA Helicases