Alternative Processing Pathways Research Articles

Two mRNAs can be produced from a single immunoglobulin μ gene by alternative RNA processing pathways

As shown in the accompanying paper, μ chains of the membrane-bound ( μ m) and secreted ( μ s) forms of IgM are encoded by two species of mRNA. Cloned cDNAs produced from the two μ mRNAs of M104E mouse myeloma tumors differ only at their 3′ ends, which encode either the μ m or μ s C terminus. In this paper, we show that both μ m and μ s mRNAs are produced from transcripts of a single μ gene. The last 187 nucleotides of μ s mRNA are derived from DNA contiguous with the 3′ end of the sequence encoding the C μ4 domain. The μ m cDNA clone does not include these 187 nucleotides, but instead contains 392 nucleotides derived from two exons located 1850 bp 3′ to the C μ4 sequence. Comparison of genomic and cDNA sequences show that in μ m mRNA, an RNA splice of 1850 nucleotides joins a site in the coding sequence at the end of C μ4 with a site at the beginning of the first membrane-specific exon. A second RNA splice of 118 nucleotides joins sequences transcribed from the first and second membrane-specific exons. The differences observed between μ m and μ s cDNAs suggest that developmental control of the site at which poly(A) is added to transcripts of the μ gene determines the relative levels of μ m or μ s chain synthesis. We discuss possible models for the control of μ gene transcripts and the significance of this form of developmentally regulated RNA processing for the evolution of eucaryotic “split genes.”

Alterations in the Processing of Rat-Liver Ribosomal RNA Caused by Cycloheximide Inhibition of Protein Synthesis

Cycloheximide given in vivo at low doses (2--5 mg/kg body weight) causes within 30 min a complete inhibition of protein synthesis in rat liver. The labelling of nuclear proteint is also strongly inhibited. Under these conditions, the amount of nucleolar 45-S pre-rRNA and its [14C]-orotate labelling remain unaffected for at least 4 h. These results show that initially the rates of synthesis and processing of 45-S pre-rRNA are not appreciably altered. On the other hand, drastic alterations in the 45-S pre-rRNA processing pathways occur at the early stages of cycloheximide action. Formation of 18-S rRNA is abolished and that of 28S rRNA is reduced to about half the level in control rats. This dichotomy in the production of the two ribosomal particles may be correlated with a block in the formation of 41-S and 21-S pre-rRNA. Generation of 36-S and 32-S pre-rRNA is still taking place, but the rate of their processing to nucleolar 28-S rRNA is decreased, thus causing the accumulation of these two pre-rRNA species. In parallel, processing of 45-S pre-rRNA to an aberrant 39-S rRNA species is markedly enhanced. The results obtained show that the channelling of nucleolar pre-rRNA along alternative processing pathways is under stringent control by the continuous supply of critical protein(s).

Confirmation of a 32S ribosomal RNA intermediate in insects

Synthesis and processing of 38S pre-RNA can be observed in maturing housefly ovaries following the injection of [ 3H]uridine into females. Subsequent injection of actinomycin D inhibits incorporation of label into precursor but allows processing through 32S, 30S, and 20S intermediates into mature 28S and 18S rRNA. The molecular weights of the pre-rRNA intermediates and products are similar to those seen under in vitro conditions. Existence of a 32S intermediate may indicate that alternate processing pathways for ribosomal RNA exist in insects.