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 A28, U25A3, [A؉U]26, or A3 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 ␤-subunit nor green fluorescent protein could be detected. However, a U25A3 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 A28 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.

Antisense Transcript and RNA Processing Alterations Suppress Instability of Polyadenylated mRNA in Chlamydomonas Chloroplasts

“…A different result was obtained for spa2, which gave a shorter product than D26pAtE with primer sets (YK12 þ aadA2/atpA59) but not from (YK03 þ BM39E), which indicated that spa2 had a deletion within trnE1. Indeed, sequencing of the spa2 PCR product revealed a 750-bp deletion beginning within trnE1 and extending into the aadA cassette promoter, as we previously reported (Komine et al, 2002). Because the trnE1 sequence is required for efficient processing at the RNase P site and, thus, for exposure of the poly(A) tail, the deletion of trnE1 sequence is likely to be the major reason for phenotypic reversion of spa2-5 (summarized in Figure 2C).…”

“…This precursor is processed intercistronically by RNase P, yielding a tRNA Glu molecule and an atpB transcript terminated by 28 adenosines. As we have shown previously (Komine et al, 2002), such atpB transcripts do not accumulate to a level detectable by filter blotting or RT-PCR because of inherent instability of 39 polyadenylated transcripts in the chloroplast. The resultant lack of ATP synthase assembly, and consequent nonphotosynthetic phenotype of D26pAtE, served as a basis for the selection of photoautotrophic revertants that might be impaired in the degradation of polyadenylated atpB transcripts.…”

“…First, the 39 UTR stem-loop was deleted to generate D26. Then, a poly(A) tract, the trnE1 gene including its upstream RNase P site, and an aadA selectable marker cassette were inserted, generating D26pAtE (Komine et al, 2002). (B) The atpB gene, poly(A) tract, and trnE1 gene are presumed to be cotranscribed into a pre-mRNA.…”

“…Exposure of the poly(A) tail further reduced mRNA stability, resulting in an obligate heterotrophic phenotype because of the absence of the ATP synthase complex. This offered an opportunity to conduct a genetic screen to identify nuclear genes or chloroplast cis-elements that were involved in polyadenylation-stimulated degradation, and initial results suggested that suppressors that accumulated atpB mRNA could be readily obtained (Komine et al, 2002).…”

An mRNA 3′ Processing Site Targets Downstream Sequences for Rapid Degradation in Chlamydomonas Chloroplasts

“…First, concomitantly with 5Ј cleavage stimulation by monophosphate groups, RNase E is responsible in part for cleaving 3Ј poly(A) tails (36). We have shown such tails to destabilize Chlamydomonas atpB mRNA both in vitro (37) and in vivo (38). Thus, an RNase E-like activity could cause rapid degradation by attacking both ends.…”