Deadenylation-Dependent and -Independent Decay Pathways for α1-Tubulin mRNA in Chlamydomonas reinhardtii

ABSTRACT

The α- and β-tubulin mRNAs of Chlamydomonas reinhardtii exhibit different half-lives under different conditions: when expressed constitutively, they degrade with half-lives of about 1 h, whereas when induced by deflagellation, they degrade with half-lives of only 10 to 15 min. To investigate the decay pathway(s) used under these two conditions, an α1-tubulin gene construct which included an insert of 30 guanidylate residues within the 3′ untranslated region was introduced into cells. This transgene was efficiently expressed in stably transformed cells, and the mRNA exhibited constitutive and postinduction half-lives like those of the α1-tubulin mRNA. Northern blot analysis revealed the occurrence of a 3′ RNA fragment derived from the poly(G)-containing α1-tubulin transcripts. The 3′ fragment was shown to accumulate as full-length mRNA disappeared in actinomycin D-treated cells, indicating a precursor-product relationship. Insertion of a second poly(G) tract upstream of the first resulted in accumulation of only a longer 3′ fragment, suggesting that the decay intermediate is generated by 5′-to-3′ exonucleolytic digestion. A translational requirement for generation of the 3′ fragment was demonstrated by experiments in which cells were deflagellated in the presence of cycloheximide. Analysis of fragment poly(A) length revealed that the fragments were, at most, oligoadenylated in nondeflagellated cells but had a long poly(A) tail in deflagellated cells. These findings suggest that the oligoadenylated fragment is a decay intermediate in a deadenylation-dependent, constitutive degradation pathway and that the requirement for deadenylation is bypassed in deflagellated cells. This represents the first example in which a single transcript has been shown to be targeted to different decay pathways under different cellular conditions.

ACKNOWLEDGMENTS

We thank John Anderson, Denise Muhlrad, and Roy Parker for advice on sequencing through poly(G) tracts and poly(C) oligonucleotide hybridizations. We are grateful to the Joel Rosenbaum laboratory for gifts of plasmids and cell strains.

This work was supported by grants from the NSF (MCB9117835) and the USDA/NRI Program (9701366). J.F.G. was supported by Public Health Service predoctoral training grant CA-09563.