Ribonucleoprotein organization of eukaryotic RNA. XXXI. Structure of the U1 small nuclear ribonucleoprotein.
A small nuclear ribonucleoprotein, U1 snRNP, has been implicated in mRNA processing. In this investigation sites of protein binding on U1 RNA were mapped by nuclease protection and RNA sequencing. Partially purified human U1 snRNP was sequentially digested with Escherichia coli RNAase III and S1 nuclease. The resistant ribonucleoprotein fragments were deproteinized, preparatively hybridized to the U1 RNA--complementary DNA strand of a human U1 gene cloned in bacteriophage M13, and displayed by electrophoresis. The nuclease-resistant U1 RNA fragments were between 23 and 63 nucleotides in length. Most of these fragments were not obtained when protein-free U1 RNA was similarly digested, whereas others were obtained in low yield from U1 RNA and much higher yield from U1 snRNP. RNA sequencing of the fragments revealed that the protein-protected sites in U1 snRNP correspond to base-paired stems I and II, loop a, and portions of stems III and IV (secondary structure nomenclature of Branlant et al., 1981). Single, "bulged" pyrimidines are present within the protein-covered helical regions of stems I and III. Most interestingly, the single-stranded 5' end of U1 RNA, implicated in mRNA splicing, was also highly protected by protein. These results demonstrate that the great majority of U1 RNA is covered by protein in U1 snRNP. The association of protein with the 5' end of U1 RNA is in agreement with recent evidence that snRNP proteins potentiate the binding of this region of U1 RNA with pre-mRNA splice sites.
D001345:Autoradiography; D001483:Base Sequence; D001665:Binding Sites; D004591:Electrophoresis, Polyacrylamide Gel; D004720:Endonucleases; D004722:Endoribonucleases; D029968:Escherichia coli Proteins; D006801:Humans; D009690:Nucleic Acid Conformation; D012313:RNA; D012342:RNA, Small Nuclear; D043244:Ribonuclease III; D012261:Ribonucleoproteins; D017411:Ribonucleoproteins, Small Nuclear; D015719:Single-Strand Specific DNA and RNA Endonucleases
D029968:Escherichia coli Proteins; D012342:RNA, Small Nuclear; D012261:Ribonucleoproteins; D017411:Ribonucleoproteins, Small Nuclear; D012313:RNA; D004720:Endonucleases; D004722:Endoribonucleases; D043244:Ribonuclease III; C468366:ribonuclease III, E coli; D015719:Single-Strand Specific DNA and RNA Endonucleases