RNA stability in terminally differentiating fibre cells of the ocular lens.
During terminal differentiation of lens fibre cells all cytoplasmic organelles are degraded abruptly. This process eliminates light-scattering elements from the optical axis of the lens and thereby ensures the transparency of the tissue. With the breakdown of the nucleus, transcription ceases, but the degree to which extant RNA is translated in the anucleated cells is uncertain. Previous studies indicated that fibre cell mRNA is unusually stable. For example, full-length delta-crystallin transcripts have been detected in core fibres months after transcription in these cells ceased. In the present study, we used the embryonic chicken lens as a model to examine the fate of RNA in the period immediately before and after organelle degradation. We mapped the tissue distribution of ribosomal RNA (rRNA) using acridine orange staining, in situ hybridization, and direct visualization of ribosomes by electron microscopy. These experiments suggested that rRNA decayed in the anucleated core fibre cells with a half-life of approximately 2.5 days. Similarly, in situ hybridization analysis of polyadenylated transcripts, beta-actin, or GAPDH mRNA indicated that these sequences were not stable in the core fibre cells. However, in agreement with earlier findings, we detected a strong in situ hybridization signal for delta-crystallin in the lens core, many days after transcription had ceased. We used quantitative PCR to compare the levels of GAPDH, L14 and delta-crystallin transcripts in the core region during development. Surprisingly, all three mRNAs decayed with indistinguishable kinetics. We conclude that the persistent delta-crystallin hybridization signal was not evidence of an unusually stable mRNA but, rather, reflected the extraordinary initial abundance of this transcript. Taken together, our data indicate that the half-life of both mRNA and the protein synthetic machinery in the lens core is only a few days. Given that, in vertebrate lenses, nuclei in this region of the lens are degraded during embryonic development, protein synthesis in central lens fibre cells is probably completed well before birth.
Faulkner-Jones
Beverly
B
Department of Ophthalmology and Visual Sciences, Washington University School of Medicine, St Louis, MO 63110, USA.
Zandy
Anna J
AJ
Bassnett
Steven
S
D000165:Acridine Orange; D000818:Animals; D002454:Cell Differentiation / Q000235:genetics; D002642:Chick Embryo; D005456:Fluorescent Dyes; D017403:In Situ Hybridization / Q000379:methods; D007908:Lens, Crystalline / Q000196:embryology*; D018613:Microscopy, Confocal / Q000379:methods; D008854:Microscopy, Electron / Q000379:methods; D020871:RNA Stability / Q000235:genetics*; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics; D012335:RNA, Ribosomal / Q000032:analysis / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D014158:Transcription, Genetic; D038207:delta-Crystallins / Q000032:analysis / Q000235:genetics
D005456:Fluorescent Dyes; D012333:RNA, Messenger; D012335:RNA, Ribosomal; D038207:delta-Crystallins; D000165:Acridine Orange