Evidence for in vivo modulation of chloroplast RNA stability by 3′-UTR homopolymeric tails in Chlamydomonas reinhardtii

Abstract

Polyadenylation of synthetic RNAs stimulates rapid degradation in vitro by using either Chlamydomonas or spinach chloroplast extracts. Here, we used Chlamydomonas chloroplast transformation to test the effects of mRNA homopolymer tails in vivo, with either the endogenous atpB gene or a version of green fluorescent protein developed for chloroplast expression as reporters. Strains were created in which, after transcription of atpB or gfp, RNase P cleavage occurred upstream of an ectopic tRNA(Glu) moiety, thereby exposing A(28), U(25)A(3), [A+U](26), or A(3) tails. Analysis of these strains showed that, as expected, polyadenylated transcripts failed to accumulate, with RNA being undetectable either by filter hybridization or reverse transcriptase–PCR. In accordance, neither the ATPase β-subunit nor green fluorescent protein could be detected. However, a U(25)A(3) tail also strongly reduced RNA accumulation relative to a control, whereas the [A+U] tail did not, which is suggestive of a degradation mechanism that does not specifically recognize poly(A), or that multiple mechanisms exist. With an A(3) tail, RNA levels decreased relative to a control with no added tail, but some RNA and protein accumulation was observed. We took advantage of the fact that the strain carrying a modified atpB gene producing an A(28) tail is an obligate heterotroph to obtain photoautotrophic revertants. Each revertant exhibited restored atpB mRNA accumulation and translation, and seemed to act by preventing poly(A) tail exposure. This suggests that the poly(A) tail is only recognized as an instability determinant when exposed at the 3′ end of a message