Nucleic Acids Research

In HeLa cells the "small nuclear" RNA has a cap II 5' structure (8) — m32,2,7G(5') pppXmpYmp — where X and Y are 2'0 methylated adenosine and uridine. In contrast hnRNA contains only cap I structures where the 2'0 methylated residue may be any base as was earlier reported for cytoplasmic mRNA (8,9,11). With a clear distinction between the source of these two caps an analysis of the size distribution of capped hnRNA could be performed which revealed over 65% of the capped hnRNA molecules were larger than cytoplasmic mRNA. INTRODUCTION Mammalian cell nuclei contain a collection of discrete small RNA molecules {snRNA, small nuclear RNA (1,2)} for which no function has yet been determined. Most of these RNAs derive from sites in DNA that are repeated a few hundred to a few thousand times (3,4). Some of the snRNA molecules have been found in association with the nucleolus while others may be present in the extranucleolar fraction or associated with chromatin. Several theories of transcriptional initiation and control have suggested possible roles, as yet unconfirmed, for the snRNA (5,6) . One particular feature of the snRNAs has attracted attention recently (7). The 5 terminus of several such species in rodent tumor cells was shown to be a blocked methylated oligonucleotide with the structure m.-2, 2, 7G (5 ') pppXmpYmp where X is A and Y is U with -CH., groups on the 2' positions of the riboses. These studies preceded the independent discovery that mRNA and hnRNA also possessed blocked 5' termini, "caps" as the structures have been termed (see 8 for review). In mRNA and hnRNA of mammalian cells, the structure is m GpppXmp or © Information Retrieval Limited 1 Falconberg Court London W1V5FG England Nucleic Acids Research m GpppXmpYrap. While the caps of hnRNA and mRNA have been reported to contain a similar distribution of all four bases in the X and Y positions, the m G has been identified by chromatographic systems that do not distinguish well between m G and 1^2,2,7G. If the snRNA were related to hnRNA either as a processed byproduct with a conjectured regulatory role (5) or as a primer from which to initiate synthesis (6) it might be suspected that some m.,2,2,7G would be present in hnRNA. With this possibility in mind we isolated the caps from snRNA, hnRNA and mRNA and found that only snRNA contained the m32,2,7G. This finding allowed a quantitative assessment of the distribution of caps in hnRNA which showed 1) a high proportion of nuclear transcripts contain a cap and 2) 65-75% of caps are in molecules larger than average mRNA size. EXPERIMENTAL PROCEDURES Labeling of cells. Suspension cultures of HeLa cells growing in Eagle's medium were labeled with (methylH) raethionine or ( P) as described (9). Cells were pretreated with 0.04 yg per ml. of Actinomycin D (10) only in the three hour label experiments. The total nuclear RNA was extracted from treated nuclei as described (9), denaturated in 95% DMSO at 37°C and sedimented in sucrose gradients in 0.05M NaCl, 0.2% SDS. Different hnRNA fractions were collected as indicated using rRNA as markers and the snRNA was obtained from the RNA sedimenting slower than 10S. This fraction was ethanol precipitated, digested with 100 yg per ml. of DNAse I, previously treated with iodoacetate, for 1 minute at 37 C and the reaction stopped by phenol extraction (1). The small RNA was fractionated by slab gel electrophoresis (40 cm length x 20 cm width) in 7% acrylamide gels, 1:20 bisacrylamide, in tris-boric acid buffer, pH, 8.3. P labeled samples were autoradiographed and the (methylH) methionine small RNA was analyzed by cutting out 0.5 cm bands and assayed after solution of the gel in Protosol. The RNA was extracted from the separated bands by suspending the acrylamide gel in the sucrose gradient buffer without sucrose and extruding the gel through a narrow needle under pressure. The acrylamide was spun down at 10,000 rpm in a Sorvall