Structural organization of ribonucleoproteins containing small nuclear RNAs from HeLa cells. Proteins interact closely with a similar structural domain of U1, U2, U4 and U5 small nuclear RNAs.

Abstract

Abstract U 1 snRNP † isolated from HeLa cells and purified by centrifugation in cesium chloride contains a set of proteins that may be resolved into four/five polypeptides by gel electrophoresis. When this particle was submitted to extensive digestion with micrococcal nuclease, RNA fragments of about 25 nucleotides in length were obtained. Sequence analyses showed that these highly protected fragments were derived from the same region of the U 1 molecule, spanning positions 119 to 143. At low concentrations of nuclease, a longer fragment, from nucleotide 119 to the 3′ OH end, was also detected. U 1 core-resistant snRNP, isolated by high performance liquid chromatography, still contains all the protein components of the intact particle. When a less drastically purified U 1 snRNP containing, beside the four/five polypeptides remaining after centrifugation in cesium chloride, a set of at least three polypeptides of larger size, was digested with the nuclease, no other protected RNA fragment was detected. When a mixture of U 1 , U 2 , U 4 , U 5 and U 6 snRNPs, which contains the same four/five polypeptides as U 1 snRNP, was treated with micrococcal nuclease, protected fragments of snRNAs U 2 , U 4 and U 5 were found in addition to those derived from U 1 . No fragment derived from U 6 was found. In all cases, the region of snRNA shielded from nuclease attack corresponds to a distinctive feature of the molecule. It is a single-stranded region, comprising the sequence A(U) n G with n ≥ 3, bordered by two double-stranded stems. One of these stems includes the 3′ terminus of the RNA, except in the case of U 2 , where there are two stems instead of one on the 3′ side of the single-stranded stretch. Although a comparable structural domain exists also in U 6 snRNA, it does not contain the sequence A(U) n G which correlates well with the fact that no U 6 snRNA fragment seems to resist micrococcal nuclease digestion.