Distribution of low-molecular-weight RNAs in avian erythroblast nuclear ribonucleoprotein complexes associated with pre-messenger RNA.

Abstract

Duck erythroblast nuclei, isolated under conditions designed to preserve lysosome integrity, were extracted to yield a bulk population of nuclear ribonucleoprotein (nRNP) particles which, when fractionated on sucrose gradients, was resolved into two classes of RNA‐protein complexes containing low‐molecular‐weight RNA. The classical pre‐messenger ribonucleoprotein complex (pre‐mRNP), which had been previously characterized in this laboratory and was shown to contain rapidly‐labeled pre‐messenger RNA (pre‐mRNA) sequences, was observed to contain approximately eight discrete species of nuclear RNA between 90 and 230 nucleotides in length (major species of Mr 80000 and 55000, and minor species of Mr 61 000, 58000, 51000, 38000, 36000 and 32000). The size and relative distribution of these RNA sequences indicated that they were the small nuclear RNA species previously described in the literature.In addition, two smaller ribonucleoprotein complexes with sedimentation coefficients of 17 S and 12 S were characterized and shown to be composed of characteristic, although not mutually exclusive, sets of low‐Mr RNA species. Comparison of these RNA species in the two smaller nRNP complexes with those found in the 40‐S pre‐mRNP particles demonstrated that all but three of the major low‐Mr RNA bands found in the small nRNP were found in the pre‐mRNP. The additional RNA species unique to the small nRNP were major ones of MI 150000 and 110000, minor ones of Mr 48000, 34000 and 27000 and three major bands of Mr approximately 25000 as well.Further comparison revealed that while each particle was composed of a characteristic subset of polypeptides, several common proteins were observed in both small nRNP and pre‐mRNP. The common RNA and polypeptide components observed in these two classes of nuclear ribo‐ nucleoprotein suggested that these small nRNPs may represent either integral parts of intact physiological pre‐mRNP or nuclear structures which are closely associated with pre‐mRNP in vivo.No difference was observed in either the RNA or protein composition of small nRNP or pre‐mRNP when prepared in the presence of the protease inhibitor phenylmethylsulfonyl fluoride, indicating that endogenous protease activity was not a problem in the preparation of nRNP from duck erythroblasts. CsCl gradient analysis of the two fractionated small nRNPs revealed buoyant densities of 1.39 and 1.40 g/ml for the 17‐S and 12‐S particles respectively. From these densities, an approximate RNA: protein ratio of 1 : 4, similar to that previously determined for the 40‐S pre‐mRNP complex, was estimated. Isoelectric focusing of the small nRNP on a pH gradient between pH 4 and pH 6 and electron microscopy studies reaffirmed the particulate nature of these smaller ribonucleoprotein complexes. In addition, ultrastructural analysis of small nRNP particles indicated an average diameter of 15 nm, compared with an average diameter of 35 ‐ 40 nm for the bulk of the 40‐S pre‐mRNP particles.The poly(A)‐protein complex of pre‐RNA, sedimenting at a position of 15 S on the sucrose gradients, was bound to oligo(dT)‐cellulose and subsequently eluted from the column in buffer containing 50 % formamide. Six major proteins were associated with this poly(A) sequence, including the 73 000‐Mr poly(A)‐binding protein typically seen associated with the poly(A) sequence of mRNA and pre‐mRNA. In contrast, neither the 17‐S nor the 12‐S nRNP was bound to the oligo(dT)‐ cellulose column indicating that these nRNPs did not contain poly(A) sequences and were, there‐ fore, nRNP complexes distinctly different from the previously described poly(A)‐protein complex of pre‐mRNA.