Comparison of the Nuclear Ribonucleoproteins Containing the Transcripts of Adenovirus‐2 and HeLa Cell DNA

Abstract

A method was devised allowing the preparation of hnRNA-containing ribonucleproteins (hnRNP) from HeLa cells infected with adenovirus type 2 under conditions where the extraction of viral replication complexes was minimal. Approximately 60 % of the RNA from such hnRNP hybridized with adenovirus DNA. The hnRNP from infected cells had the same general characteristics as those from uninfected cells. Their size was heterogeneous (30-260 S) and depended upon that of their RNA. Their CsCl densities were identical (1.39-1.40 g/ml), indicating the same protein:RNA ratio. Their proteins were found in the same molecular weight range, between 25 000 and 200000.The major proteins of hnRNP from HeLa cells were present in hnRNP from adenovirusinfected cells. As 60 % of the cellular RNA was replaced by adenovirus RNA in hnRNP, this indicated that there was not stringent specificity in the RNA-protein interactions. The relative proportions of the proteins were identical in both cases, suggesting that the hnRNP assembly was independent of nucleotide sequences at least for the major proteins. The hnRNP from infected and uninfected cells differed by the size of their RNA, which was larger after infection, and by the presence of six additional minor polypeptides after infection. However, it cannot be excluded that the presence of these polypeptides in hnRNP resulted from non-specific adsorption.hnRNA-containing ribonucleoproteins (hnRNP) are assumed to be the site for the processing of the precursors of messenger RNA. It seems surprising that only few attempts have been made, up to now, to study the hnRNP containing the transcripts of the adenovirus genome [I -31 though much information on the synthesis of the viral RNA is already available [4-81 and though the first demonstration of splicing was achieved in that system [9]. These considerations prompted us to start a study of hnRNP containing adenovirus-2 RNA. Our first aim was to demonstrate whether such hnRNP were similar to cellular hnRNP or whether they presented specific features. Therefore, we choose to work at a period post-infection (15 h) where a relatively large proportion of the nuclear RNA was of viral origin [lo]. The first step involved elaboration of a method for preparing hnRNP from adenovirus-infected cells. In particular, the current methods had to be modified in order to reduce the proportion of viral DNA-containing complexes which were extracted together with the hnRNP. Then, we demonstrated that viral RNA was Ahhreviations. hnRNA, heterogeneous nuclear RNA; hnRNP, ribonucleoproteins containing hnRNA; mRNP, ribonucleoproteins containing messenger RNA. the major RNA from these hnRNP and we compared the characteristics of hnRNP from adenovirus-infected cells with those from uninfected HeLa cells. Our main conclusion is that the two classes of hnRNP are similar, showing the absence of stringent specificity of the RNA-protein interactions. MATERIALS AND METHODS Cell Culture and VirusHeLa cells were grown in suspension, infected with adenovirus-2 (100 plaque-forming ...

An evaluation of small nuclear RNA in hnRNP

“…On an average, 50% of the snRNA were found in each nuclear fractions. As a relatively important fraction of hnRNP remained bound to chromatin under our experimental conditions [23], it is likely that 50% is an underestimation. The electrophoretic profiles of the 2 fractions were close, with the exception of the presence of U3 RNA and of 1 small band between Ul RNA and the major 5 S RNA preferentially localized in the pellet.…”

The orderly splicing of the first three leaders of the adenovirus-2 major late transcript

“…In order to eliminate variations due to the labeling kinetics of the nucleotide pool and of the primary transcripts, and to study only the splicing reactions, we used RNA samples with identical amounts of radioactivity for each hybridization reaction. The amount of radioactive viral RNA in the samples was henceforth identical, since the distribution of the label between cellular and viral RNA does not change up to 120 min (15,34). The possible interferences due to widely different RNA amounts were eliminated by using adequate amounts of [3H]-uridine for labeling as described under Material and Methods.…”

“…Labeling was with [3H]-uridine (10 Ci/mmol) at a cell concentration of 6 x 106 cells/ml (15) in the last minutes of infection. In order to obtain equivalent specific activities of nuclear RNA in the kinetics experiments, various amounts of [3H]-uridine were used : 500 pCi/ml (10 min), 300 pCi/ml ( (20).…”

Modulation of alternative splicing of adenoviral E1A transcripts: factors involved in the early-to-late transition.

“…We have shown that the inducing nucleic acids are RNAs of high molecular weight and that the inducing ability originates mainly, if not exclusively, from viral RNAs, most probably synthesized from the major late transcription unit. This is not unexpected, because late in infection the rate of transcription of this unit increases dramatically; and at this stage up to 50-60% of the nonribosomal nuclear RNA are of viral origin (Manley et al 1979;Gattoni et al 1980;Shaw and Ziff 1980). The accumulation of viral transcripts in the form of unspliced and partially processed molecules (Ziff 1980 and references therein) may result in dramatic consequences for cellular processes that may be rapidly saturated.…”