Polysome-bound RNA Research Articles

Translation in vivo of 5′ untranslated-region splice variants of human surfactant protein-A

Transcripts of human SP-A genes, SP-A1 and SP-A2, undergo alternative splicing of 5' untranslated-region exons. We reverse-transcribed and amplified free cytoplasmic and polysome-bound RNA and showed that (a) all splice variants of both genes are translated in vivo, (b) the relative translatability of splice variants can differ among individuals, and (c) the relative levels of different SP-A splice variants differ among individuals.

Mouse opsin. Gene structure and molecular basis of multiple transcripts

The single copy mouse opsin gene produces five major transcripts, varying in size from 1.7 to 5.1 kilobases. The mRNAs are present at levels that vary over 2 orders of magnitude and can be detected as early as postnatal day 1. Each of the transcripts is polyadenylated and can be identified in polysome-bound RNA, suggesting that each is translated in vivo. To elucidate the molecular basis of this complex transcription pattern, we have characterized genomic fragments covering the entire mouse opsin gene, including several kilobases of 5'- and 3'-flanking regions. Transcription initiates at a single site 97 base pairs upstream of the translation start codon. Northern hybridization with exon- and intron-specific probes demonstrated that the various transcripts are not generated by partial or alternative splicing. Sequence analysis of the 3' end of the gene showed the presence of multiple polyadenylation signals. Analysis by polymerase chain reaction of the 3' end of opsin cDNA demonstrated that the complex transcription pattern originated from the selective use of these polyadenylation sites, generating transcripts that differ only in the length of the 3'-untranslated region. Transcript heterogeneity similar to that observed in mouse was also found in rat and, to a lesser degree, in human and frog opsin mRNAs.

In vitro synthesis, importation and processing of Mn-superoxide dismutase (SOD-3) into maize mitochondria

In vitro translation of maize scutellar polysome-bound RNA and poly(A) + RNA produces a precursor for the mitochondrial superoxide dismutase (SOD-3) of maize, which can be immunoprecipitated with SOD-3 monospecific antibodies. The precursor SOD-3 (preSOD-3) has both a greater molecular weight and a more positive isoelectric point than the mature (purified) SOD-3. These differences were not observed for the cytosolic isozyme (SOD-4) which was similarly synthesized and isolated. PreSOD-3 specifically associates with maize mitochondria when incubated in vitro, whereas SOD-4 does not. the ionophore valinomycin (at concentrations of over 1 μM) inhibits the uptake of preSOD-3 into mitochondria. The integrity of the mitochondrial preparations was determined by assay of oxygen consumption, citrate synthase, and cytochrome- c oxidase activity.

Decreased protein synthesis by microsomes from aging Drosophila melanogester

A microsomal preparation from larvae of Drosophila melanogaster readily incorporated a 14C-amino acid mixture into its protein. Amino acid incorporation by microsomes from adult organisms, however, declined markedly with age. The decline in amino acid incorporation did not appear to be due to decreases in ribosomes or messenger RNA, since ribosome levels decreased only 6%, while incorporation decreased more than 70%, and only a 23% decrease in polysome-bound RNA could be detected. However, the ppolyuridylate-promoted incorporation of 14C-phenylalanine into polypeptide exhibited a sharp, age-related decline, indicating that some component of the translation system, other than messenger RNA, is responsible for the observed decline in protein synthesis