Loss of the polyadenylate segment from mammalian messenger RNA. Selective cleavage of this sequence from polyribosomes

BERGMANN, I.E.; BRAWERMAN, G.

JOURNAL OF MOLECULAR BIOLOGY

Año: 1980 vol. 139 p. 439 - 454

0022-2836

Treatment of mouse sarcoma 180 ascites cell polysomes with low levels of micrococcal nuclease, under conditions that cause relatively little fragmentation of the messenger RNA chains, results in considerable loss of poly(A) from these chains. This treatment generates a substantial amount of functional poly(A)-lacking mRNA. Brief incubation of cytoplasmic extracts of the ascites cells, and of mouse liver extracts, has similar effects on the polysomes present in the extracts and on the generation of poly(A)-lacking mRNA chains. The poly(A) segment is released from the polysomes treated with micrococcal nuclease as a nucleoprotein complex, and is protected from the action of the enzyme because of its association with protein. There is considerable poly(A) hydrolysis in incubated ascites cell extracts, and accumulation of a poly(A)-protein complex does not take place in this case. The liver extracts have little poly(A)-hydrolyzing activity, and free poly(A)-protein complexes are observed in these extracts. The poly(A)-cleavage process shows evidence of considerable selectivity. The newly synthesized mRNA population is more susceptible to this process than is the steady-state population. Moreover, only a portion of the steady-state mRNA loses its poly(A) readily upon incubation with micrococcal nuclease. Two-dimensional gel electrophoresis of translation products from total and poly(A)-lacking polysomal RNA preparations shows that not all mRNA species lose their poly(A) upon incubation of polysomes in ascites cell extracts. The sensitive population resembles the normal population of translatable poly (A)-lacking mRNA that is obtained from untreated polysomes. Individual species within this population show wide differences in their degree of susceptibility to the poly(A)-release process in vitro. Analysis by one-dimensional gel electrophoresis indicates that the same general population is generated by the incubation of cytoplasmic extracts and by the treatment of polysomes with micrococcal nuclease. It is suggested that the 3Œ non-coding region of mRNA in polysomes is particularly sensitive to endonucleolytic cleavage, and that loss of poly(A) via this cleavage may be a normal cellular process. The diversity in nucleotide sequence and in overall configuration in this region could provide a basis for the observed differences in susceptibility to cleavage by nucleases.