Methylphosphate cap structure in small RNAs reduces the affinity of RNAs to La protein.
La protein is an abundant 47-kDa phosphoprotein found mostly in the nucleus of eukaryotic cells with a small fraction present in the cytoplasm. Nascent RNA transcripts synthesized by RNA polymerase III are known to be associated with La protein. This binding has been shown to occur to the 3' end of RNA via RNA recognition motifs and to the 5' triphosphate via the Walker A motif of the La protein. In this study, we developed an in vitro immunoprecipitation assay to quantitate the 5' ppp-dependent binding of small RNAs to the human La protein. Using this assay, we found that oligonucleotides five bases or longer bind to the human La protein in a 5' ppp-dependent manner, pppG did not bind to La protein in this assay. In addition, CH3pppN cap structure present on the 5' ends of U6 and B2 small RNAs reduced the ability of these RNAs to bind the human La protein. These data show that Walker motif in the human La protein can bind to short RNAs containing 5' ppp and removal of 5' ppp from RNAs, or modification of 5' pppN to CH3pppN or m7GpppN, significantly reduces the ability of small RNAs to bind the human La protein. These data suggest that one of the functions of methylphosphate cap structure in U6 snRNA and B2 RNAs is possibly to reduce the affinity of these RNAs to La protein.
Bhattacharya
Rajat
R
Department of Pharmacology, Baylor College of Medicine, Houston, TX 77030, USA.
Perumal
Karthika
K
Sinha
Krishna
K
Maraia
Richard
R
Reddy
Ram
R
D020816:Amino Acid Motifs; D001324:Autoantigens; D053487:DEAD-box RNA Helicases; D006367:HeLa Cells; D006801:Humans; D010755:Organophosphates / Q000737:chemistry*; D011233:Precipitin Tests; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012313:RNA / Q000737:chemistry* / Q000378:metabolism*; D020365:RNA Helicases / Q000378:metabolism; D012342:RNA, Small Nuclear / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D012261:Ribonucleoproteins / Q000378:metabolism*; D000097585:SS-B Antigen
D001324:Autoantigens; D053487:DEAD-box RNA Helicases; D010755:Organophosphates; D012313:RNA; D020365:RNA Helicases; D012342:RNA, Small Nuclear; D011994:Recombinant Proteins; D012261:Ribonucleoproteins; D000097585:SS-B Antigen; C405486:U6 small nuclear RNA; C027265:methylphosphate; C448980:DDX50 protein, human