Bacillus subtilis RNase J1 endonuclease and 5' exonuclease activities in the turnover of DeltaermC mRNA.
RNase J1, a ribonuclease with 5' exonuclease and endonuclease activities, is an important factor in Bacillus subtilis mRNA decay. A model for RNase J1 endonuclease activity in mRNA turnover has RNase J1 binding to the 5' end and tracking to a target site downstream, where it makes a decay-initiating cleavage. The upstream fragment from this cleavage is degraded by 3' exonucleases; the downstream fragment is degraded by RNase J1 5' exonuclease activity. Previously, DeltaermC mRNA was used to show 5'-end dependence of mRNA turnover. Here we used DeltaermC mRNA to probe RNase J1-dependent degradation, and the results were consistent with aspects of the model. DeltaermC mRNA showed increased stability in a mutant strain that contained a reduced level of RNase J1. In agreement with the tracking concept, insertion of a strong stem-loop structure at +65 resulted in increased stability. Weakening this stem-loop structure resulted in reversion to wild-type stability. RNA fragments containing the 3' end were detected in a strain with reduced RNase J1 expression, but were undetectable in the wild type. The 5' ends of these fragments mapped to the upstream side of predicted stem-loop structures, consistent with an impediment to RNase J1 5' exonuclease processivity. A DeltaermC mRNA deletion analysis suggested that decay-initiating endonuclease cleavage could occur at several sites near the 3' end. However, even in the absence of these sites, stability was further increased in a strain with reduced RNase J1, suggesting alternate pathways for decay that could include exonucleolytic decay from the 5' end.
Department of Pharmacology and Systems Therapeutics, Mount Sinai School of Medicine of New York University, New York, New York 10029, USA.
Shiyi
S
Yao
Josh S
JS
Sharp
David H
DH
Bechhofer
D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000201:enzymology*; D001483:Base Sequence; D017353:Gene Deletion; D008780:Methyltransferases / Q000737:chemistry / Q000235:genetics*; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D020871:RNA Stability; D012333:RNA, Messenger / Q000737:chemistry / Q000378:metabolism*; D012260:Ribonucleases / Q000378:metabolism*; D012270:Ribosomes / Q000378:metabolism
D012333:RNA, Messenger; D008780:Methyltransferases; C035181:rRNA (adenosine-O-2'-)methyltransferase; D012260:Ribonucleases