3′-Processed mRNA Is Preferentially Translated in Chlamydomonas reinhardtii Chloroplasts

Abstract

3-end processing of nucleus-encoded mRNAs includes the addition of a poly(A) tail that is important for translation initiation. Since the vast majority of chloroplast mRNAs acquire their 3 termini by processing yet are not polyadenylated, we asked whether 3 end maturation plays a role in chloroplast translation. A general characteristic of the 3 untranslated regions of chloroplast mRNAs is an inverted repeat (IR) sequence that can fold into a stem-loop structure. These stem-loops and their flanking sequences serve as RNA 3-end formation signals. Deletion of the Chlamydomonas chloroplast atpB 3 IR in strain ⌬26 results in reduced accumulation of atpB transcripts and the chloroplast ATPase ␤-subunit, leading to weakly photosynthetic growth. Of the residual atpB mRNA in ⌬26, approximately 1% accumulates as a discrete RNA of wild-type size, while the remainder is heterogeneous in length due to the lack of normal 3 end maturation. In this work, we have analyzed whether these unprocessed atpB transcripts are actively translated in vivo. We found that only the minority population of discrete transcripts of wild-type size is associated with polysomes and thus accounts for the ATPase ␤-subunit which accumulates in ⌬26. Analysis of chloroplast rbcL mRNA revealed that transcripts extending beyond the mature 3 end were not polysome associated. These results suggest that 3-end processing of chloroplast mRNA is required for or strongly stimulates its translation.Chloroplast genes are often organized into operons and gene clusters, which are transcribed into precursor transcripts that undergo complex processing events including splicing and intercistronic cleavages (reviewed in references 35 and 49). While intercistronic cleavages form some mRNA 5Ј and 3Ј termini, these can also be formed by other types of events. For example, 5Ј ends are often formed by endonucleolytic processing of primary transcripts, and this may be the exclusive mode of 5Ј end formation in chloroplasts in the green alga Chlamydomonas reinhardtii (reviewed in reference 12). Most plastid mRNAs contain inverted-repeat (IR) sequences in their 3Ј untranslated regions, which are believed to fold into stem-loop structures. These IR sequences do not function as efficient transcription terminators but instead are thought to stabilize upstream sequences and mediate correct 3Ј-end processing (36,37,44,47,48). In most cases, the 3Ј termini of mature transcripts lie immediately downstream of the IR.Plastid 3Ј IR sequences act to stabilize upstream mRNA segments in vitro and in vivo. When RNA molecules containing the IR sequences were incubated in spinach chloroplast protein extracts, they were correctly processed at their 3Ј ends and the products were stable for several hours. However, when the IR sequences were deleted from the same RNA molecules and incubated in an identical protein extract, the RNA molecules were rapidly degraded (17,37,44,46). The ability to introduce altered genes into the chloroplast of the green alga C. reinhardtii presented the opportunity to ...

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

“…On the other hand, ⌬26AUtE accumulated Ϸ5% of the wild-type ␤-subunit level, but approximately 60% of the wild-type RNA level. One possibility is that the 3Ј-UTR itself may affect translation; we have shown a correlation between atpB 3Ј processing and translation (25). However, the RT-PCR used here does not prove that the target transcript is intact and translatable.…”

“…These modified trnE genes were inserted as BglII-BamHI fragments into the BglII sites of atpB⌬19AD, atpB⌬21AD, and atpB⌬26AD [ Fig. 1 A and B; only the (A) 25 cassette was inserted into atpB⌬19AD and atpB⌬21AD]. For GFP constructs, plasmid MR220 containing cpgfp1 (GenBank accession no.…”

“…The trnE gene was cloned by using PCR, with the amplified fragment containing a BamHI site plus 3 bp upstream of the mature tRNA sequence, and 148 bp downstream plus a BglII site. Modified trnE genes were created by PCR with an upstream primer containing a BglII site and one of following: (A) 3 , (A) 25 , (T) 25 , or the arbitrary [AϩU] 26 sequence (Fig. 1B), immediately upstream of the RNase P site; and a downstream primer containing a BamHI site.…”

“…Our studies with Chlamydomonas have detected polyadenylated forms of mRNA, tRNA, and rRNA by RT-PCR, and shown that an A 25 tail is sufficient to confer marked instability in vitro (9). Here, we have manipulated the endogenous atpB gene, which encodes the ␤-subunit of ATP synthase, and a green fluorescent protein (GFP) reporter gene, to examine the consequences of adding specific 3Ј end tails in vivo.…”

The 3′-Untranslated Region of Chloroplast psbA mRNA Stabilizes Binding of Regulatory Proteins to the Leader of the Message

“…Identification of interactions between the two termini of cytoplasmic transcripts (18,19) and examples of 3Ј-UTR modulation of translation initiation in both eukaryotes (18,20) and prokaryotes (21)(22)(23) raise the possibility that interactions of the 5Ј-and 3Ј-UTR of chloroplast mRNAs may influence their expression. In support of this notion, recent results have shown that correct processing of the 3Ј-UTR can promote translation initiation and polysomal association (24).…”