Relationship of chain transcription to poly(A) addition and processing of hnRNA in HeLa cells

Abstract

Two pathways for addition of poly(A) to hnRNA can be identified by using very brief periods of exposure to radioactive nucleosides to label hnRNA. Poly(A)-containing molecules of various size classes have been analyzed from cells exposed for 2.5 min or less to 3H-adenosine [which labels both the transcribed region of hnRNA and the poly(A) added after transcription] or to 3H-uridine (which labels only the transcribed region). After labeling for 30 sec or 2.5 min with 3H-U, more than 80% of the radioactivity that enters poly(A)-containing hnRNA is located in molecules sedimenting faster than 32S. Since incorporation of 3H-U identifies newly synthesized molecules, poly(A) must be added to some large “new” transcripts within 30 sec of exposure to 3H-U. Although only 10–15% of the poly(A)-containing molecules labeled with 3H-U sediment at less than 32S, about 35% of the molecules labeled with 3H-A for 30 sec are smaller than 32S. Most (up to 80%) of the label incorporated into small poly(A)-containing hnRNA molecules during a 1 min exposure to 3H-A is located in the poly(A) region. Since few of the small molecules are labeled with 3H-U, they must represent “old” transcripts not yet modified or fragments of originally larger transcripts to which poly(A) is added. Treatment with DMSO of hnRNA molecules sedimenting at less than 32S does not alter their properties. But DMSO treatment of hnRNA sedimenting faster than 45S appears to release small poly(A)-containing molecules. This suggests that some of the large hnRNA molecules may be partially processed, presumably maintained by secondary structure, so that on denaturation they release smaller molecules. There seems therefore to be no single program for hnRNA synthesis and processing; some (mostly large) molecules have poly(A) added immediately after synthesis, whereas other (mostly small) molecules undergo addition at a later time. It is likely also that some molecules are processed prior to addition of poly(A), whereas others are processed after addition of poly(A).