Mild nuclease treatment as a probe for a non-random distribution of adenovirus-specific RNA sequences and of cellular RNA in nuclear ribonucleoprotein fibrils

Abstract

At the late period of productive infection, the RNA of hnRNP † † Abbreviations used: hnRNA, heterogeneous nuclear RNA: premRNA, premessenger RNA; RNP, ribonucleoprotein; hnRNP, ribonucleoprotein containing hnRNA; Ad2, adenovirus type 2; EGTA. ethyleneglycol-bis(aminoethyl ether) tetraacetic acid. from adenovirus-infected HeLa cells contains primarily cellular RNA and the partially processed transcripts from the major late transcription unit. Total cellular and viral RNA, seven viral RNA fragments corresponding to messenger and non-messenger RNA sequences, were determined in hnRNP. A mild nuclease treatment of 30 to 200 S native hnRNP, sufficient for the separation of 25 to 55 S monoparticles from larger (100 to 200 S) RNP, served as a probe for hnRNP structure and allowed the study of sequence distribution in these two classes of hnRNP units. The main observations were as follows: (1) 90% of the total viral RNA initially present in large hnRNP was found in the large RNP after nuclease treatment, whereas 85% of the nuclear-restricted cellular RNA was released with monoparticles; (2) viral poly(A) + RNA was enriched in large RNP indicating an enrichment in viral premessenger RNA; (3) the viral-specific sequences were distributed non-randomly between the hnRNP units. This distribution did not depend on their nucleotide sequence (messenger or non-messenger nature). Rather, the sequences from the 5′ half of the transcription unit were enriched in large RNP and those from the 3′ half in monoparticles. This may be related to the presence, in addition to authentic premessenger RNA, of nuclear-restricted hnRNA synthesized from the 3′ part of the transcription unit after the polyadenylation sites (Nevins & Darnell, 1978). The preferential localization of premessenger RNA in the large RNP isolated after mild nuclease treatment suggested that these structures are the sites of splicing. The monoparticles that are enriched in nuclear-restricted sequences might be structures where degradation starts.