Testis brain ribonucleic acid-binding protein/translin possesses both single-stranded and double-stranded ribonuclease activities.
RNA interference (RNAi) is a biological process in which animal and plant cells destroy double-stranded RNA (dsRNA) and consequently the mRNA that shares sequence homology to the dsRNA. Although it is known that the enzyme Dicer is responsible for the digestion of dsRNA into approximately 22 bp fragments, the mechanism through which these fragments are associated with the RNA-induced silencing complex (RISC) is mostly unknown. To find protein components in RISC that interact with the approximately 22 bp fragment, we synthesized a (32)P- and photoaffinity moiety-labeled 22 bp dsRNA fragment and used it as bait to fish out protein(s) directly interacting with the dsRNA fragment. One of the proteins that we discovered by mass spectrometric analysis was TB-RBP/translin. Further analysis of this DNA/RNA binding protein showed that it possesses both ssRNase and dsRNase activities but not DNase activity. The protein processes long dsRNA mainly into approximately 25 bp fragments by binding to the open ends of dsRNA and cutting it with almost no turnover due to its high affinity toward the products. The activity requires physiological ionic strength. However, with single-stranded RNA as substrate, the digestion appeared to be more complete. Both ssRNase and dsRNase activities are inhibited by high levels of common RNase inhibitors. Interestingly, both activities can be enhanced greatly by EDTA.
Wang
Jun
J
Laboratory of Biochemistry, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892-8012, USA.
Boja
Emily S
ES
Oubrahim
Hammou
H
Chock
P Boon
PB
D000595:Amino Acid Sequence; D000818:Animals; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D019286:Endopeptidase K / Q000737:chemistry; D015723:Gene Library; D005822:Genetic Vectors; D008297:Male; D051379:Mice; D008969:Molecular Sequence Data; D041681:NIH 3T3 Cells; D011485:Protein Binding / Q000235:genetics; D012330:RNA, Double-Stranded / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D034743:RNA-Induced Silencing Complex / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D013737:Testis
D004268:DNA-Binding Proteins; D012330:RNA, Double-Stranded; D034741:RNA, Small Interfering; D016601:RNA-Binding Proteins; D034743:RNA-Induced Silencing Complex; C498303:Tsn protein, mouse; D004247:DNA; D019286:Endopeptidase K