THE PATTERN OF METHYLATION OF RNA IN PERIPHERAL NERVE OF THE CHICK DURING DEVELOPMENT¹

Abstract

The pattern of the methylation of RNA was investigated in organ cultures of the sciatic nerve of the chicken. Nerve tissue from 14‐day embryos, 17‐day embryos and 3‐day‐ old chicks was incubated with [methyl‐3H]methionine or with [2‐14C]uridine and [methyl‐3H]methionine simultaneously for various periods of time. Subsequently, RNA was extracted from the tissues and the purified preparations were fractionated by polyacrylamide gel electrophoresis. The electrophoretic patterns of the rapidly labelled RNA changed during the three developmental stages. The incorporation of both uridine and the methyl groups from methionine was highest in the‘heavy’RNA species of the 14‐day embryonic nerve during the 0.5 and 1.0 h incubation periods. In contrast, in the nerves of 3‐day‐old chicks during a 0.5 h pulse with both precursors, methylation was almost entirely limited to the transfer RNA species. Furthermore, the incorporation of uridine in the nerves from 3‐day‐old animals revealed the presence of a heterogeneous population of rapidlylabelled, unmethylated species of RNA, most of which migrated between the smaller ribosomal RNA and transfer RNA components of the bulk RNA. The pattern of uridine incorporation and the methylation of the rapidly‐labelled RNA of the 17‐day embryonic nerve represented a transitional state between that of the 14‐day embryos and that of the 3‐day‐old chicks. The 17‐day embryonic stage of development corresponded to the phase of the onset of rapid deposition of myelin lipids in the sciatic nerve. Pulse‐chase experiments on the embryonic nerves indicated that a number of methylated precursors of ribosomal RNA and labile, heterogeneous, probably DNA‐like RNA were synthesized.

Studies on Polyadenylic Acid‐Containing RNA from Developing Nervous System of the Chicken the

“…Thus, tissue of one developmental stage exposed to tissue of another stage still showed the characteristic shift in the apparent size distribution of poly(A)+ RNA. The change in the size of the poly(A)+ RNA as well as in the electrophoretic properties of crude rapidly labeled RNA of the chick nervous system during development is in agreement with previous studies done on sciatic nerve Mezei and Hu, 1972). Berthold and Lim (1976b) reported that the nuclear and cytoplasmic polyadenylated RNA species of young rat brain are also larger than their corresponding adult counterparts.…”

“…In addition, great caution should be exercised in interpreting data concerning differential synthesis of various RNA species in the absence of specific knowledge about the behavior of their precursor With the availability of affinity chromatography for the isolation of poly(A)+ RNA it has become possible to compare the polyacrylamide gel electrophoretic patterns of this RNA with those obtained previously for total rapidly labeled RNA of the identical developmental stage. Thus, if a sizable amount of poly(A)+ RNA could be obtained from embryonic and post-hatching tissues, and if the average size distribution of the poly(A)+ RNA was similar to that found previously for the crude rapidly labeled RNA of sciatic nerve and brain of chicks Mezei and Hu, 1972), one could conclude that at least part of the rapidly labeled RNA observed in these tissues was poly(A)-containing mRNA or hnRNA. The results of these investigations have borne out these expectations (Table 3).…”

“…Fertile eggs were obtained from Lone Pine Ltd. (Berwick, Nova Scotia, Canada) and incubated. Animals were maintained as described previously (Mezei and Hu, 1972 tham, Massachusetts). High-purity acrylamide, bisacrylamide, ammonium persulfate, and N , N , N ' N 'tetramethylethylenediamine were obtained from Bio-Rad Laboratories (Richmond, California).…”

“…Extraction of RNA was by a method similar to one described previously (Mezei and Hu, 1972) with some modifications to improve the yield of poly(A)+ RNA (Brawerman et al, 1972). The tissue was thawed and then homogenized at 4°C for 2 min at 2500 r.p.m.…”

“…the Previous studies from our laboratory have demonstrated that the sedimentation and electrophoretic properties of the rapidly labeled RNA of chick sciatic nerve change during development Mezei and Hu, 1972), particularly around the 17th day of embryonic life, during the onset of rapid myelination of peripheral nerve. On the basis of those results we considered that the rapidly labeled RNA might represent different populations of mRNA of these cells.…”