Binding of Xenopus oocyte masking proteins to mRNA sequences.
It has been shown previously that maternal mRNA, synthesized and stored in growing oocytes, is stabilized and blocked from translation through various mechanisms including restricted polyadenylation and the binding of proteins to 3' regulatory elements. In addition to binding sequence-specific proteins, the bulk of stored mRNA is packaged with a set of 'masking' proteins, the most abundant of which are the phosphoproteins pp56 and pp60. In this report these proteins are shown to be bound to heterogeneous mRNA sequences and not to the 3' poly(A) tract. Crosslinking studies demonstrate that all of the pp56/60 present makes direct contact with the RNA. In vitro binding studies confirm that pp56/60 interact with single-stranded RNA of heterogeneous sequence, such as occurring in the maternal mRNA encoding cyclin B1. However, binding is equally effective to capped and polyadenylated cyclin mRNA, to truncated mRNA lacking 5' and 3' non-coding regions and even to the antisense sequence. Lengths of 70-80 nucleotides are protected from ribonuclease digestion after protein binding. Although no extended binding motif could be detected, binding does appear to have some specificity in that it is not competed out by 100-fold excess of double-stranded RNA, transfer RNA, poly(A) and various other homopolymers and heteropolymers. The sequence which competes most efficiently is the mixed polypyrimidine, poly(C,U). Crosslinking of RNA-protein complexes, followed by ribonuclease digestion, suggests that the arrangement of proteins on RNA is as dimers. Dimerization appears to be stabilized by phosphorylation of pp56/60. These results are discussed in terms of the known structures of pp56/60.
Marello
K
K
School of Biological and Medical Sciences, University of St Andrews, Fife, UK.
LaRovere
J
J
Sommerville
J
J
D000818:Animals; D001483:Base Sequence; D004247:DNA; D004591:Electrophoresis, Polyacrylamide Gel; D008969:Molecular Sequence Data; D009865:Oocytes / Q000378:metabolism; D011485:Protein Binding; D012333:RNA, Messenger / Q000138:chemical synthesis / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000378:metabolism*; D012261:Ribonucleoproteins / Q000737:chemistry / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism*; D029867:Xenopus Proteins; D014982:Xenopus laevis
D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D012261:Ribonucleoproteins; D014157:Transcription Factors; D029867:Xenopus Proteins; C075593:YBX2 protein, Xenopus; C026452:messenger ribonucleoprotein; D004247:DNA