petD mRNA maturation in Chlamydomonas reinhardtii chloroplasts: role of 5' endonucleolytic processing.

Abstract

Complex processing of primary transcripts occurs during the expression of higher-plant chloroplast genes. In Chlamydomonas reinhardtii, most chloroplast genes appear to possess their own promoters, rather than being transcribed as part of multicistronic operons. By generating specific deletion mutants, we show that petD, which encodes subunit IV of the cytochrome b6lf complex, has an RNA processing site that is required for accumulation of monocistronic petD mRNA in petD promoter deletion mutants; in such mutants, transcription ofpetD originates from the upstream petA promoter. The 5' ends of transcripts initiated at the petD promoter are probably also generated by processing, since the 5' end of monocistronic petD mRNA is the same in wild-type strains as it is in the petD promoter mutants. The location and function of the processing site were further examined by inserting petD-uidAl fusion genes into the chloroplast genome (uid4 is an Escherichia coli gene that encodes ,-glucuronidase). When a promoterless petD-uidA fusion gene was inserted downstream of petA, a monocistronic uid4 transcript accumulated, which was apparently initiated at the petA promoter and was processed at a site corresponding precisely to the petD mRNA 5' end. When a construct including only sequences downstream of +25 relative to the mature mRNA 5' end was inserted into the same site, a dicistronic petA-uidA4 transcript accumulated but no monocistronic uid4 transcript could be detected, suggesting that a processing site lies at least partially within the region from -1 to +25. ,1-Glucuronidase activity was not detected in transformants that accumulated only the dicistronic petA-uid4 transcript, suggesting that the first 25 bp of the 5' untranslated region are required for translation initiation. One explanation for this translational defect is that Chlamydomonas chloroplasts cannot translate the second coding region of some dicistronic messages.Higher-plant chloroplast genes are organized as operons that are transcribed into polycistronic primary transcripts. Splicing and other endo-and exonucleolytic cleavages subsequently occur to generate mature transcripts (1,9,12,32). One such operon in higher plants contains the petD gene, which encodes subunit IV of the cytochrome bdlf complex. Its genomic location is conserved in higher plants (13,30) and cyanobacteria (15); however, its mode of expression varies. In spinach chloroplasts, a primary transcript containing the psbB, psbH, petB, and petD coding regions is processed into 18 RNA species, including monocistronicpsbB andpsbH transcripts and a dicistronic petB-petD transcript (32). As no monocistronic petD mRNA has been detected in spinach chloroplasts, the coding region is presumed to be translated from the dicistronic message. The transcription ofpetD RNA in maize chloroplasts also occurs as part of the psbB operon, but the major translatable form of the petD message is monocistronic. Nonetheless, polycistronic transcripts can be immunoprecipitated from maize polysomes with subunit IV-spe...

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

“…One reason for its efficiency is that positions more than 1 nt upstream of prokaryotic RNase P sites do not contribute significantly to catalytic rate or site recognition (29). This contrasts with some other endonuclease cleavage sites; for example, the petD mRNA 5Ј-processing site is used inefficiently in ectopic locations (30,31). Another advan- 6.…”

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

“…3Ј 3 5Ј Exonuclease (ii) is known from 3Ј processing studies (14) and can also be impeded by pG or by a stem-loop. Finally, endonuclease activities exist (iii), for example those which mature the 5Ј end of petD mRNA (33) or cleave at the atpB 3Ј ECS.…”

In vivo evidence for 5′→3′ exoribonuclease degradation of an unstable chloroplast mRNA

“…∆P is photosynthetic and accumulates high levels of the monocistronic petD transcript, while FUD6 does not accumulate monocistronic petD mRNA and is nonphotosynthetic. As shown in Figure 4(b), tetrad progeny from the cross ∆P ϫ F16 segregated 2:2 for accumulation of the monocistronic petD transcript and, as expected, all progeny of the cross FUD6 ϫ F16 failed to accumulate monocistronic petD mRNA (Sakamoto et al, 1994b). However, all progeny of both the ∆P ϫ F16 and FUD6 ϫ F16 crosses accumulated low levels of the 4.4 kb dicistronic petA-petD transcript (Figure 4b).…”

“…We have previously described mechanisms by which the 5Ј end of the petD transcripts may be generated (Sakamoto et al, 1994b), and we have shown that the petD 3Ј end can be correctly processed in vitro (R. Rott et al, unpublished data) by a mechanism which may resemble the endonuclease-exonuclease processing of the 3Ј ends of Chlamydomonas chloroplast atpB mRNA and the spinach chloroplast petD transcript . Briefly, these models propose that the petD 5Ј end is produced by endonucleolytic cleavage of a precursor emanating from either the petD promoter or the upstream petA promoter, and that the 3Ј end is generated by cleavage downstream of a 3Ј stem-loop followed by exonuclease resection.…”

“…The deletions in these strains, ∆P and FUD6, have been described previously (Sakamoto et al, 1994b;Sturm et al, 1994), and are shown in Figure 4(a). In both ∆P and FUD6, a 4.4 kb petA-petD cotranscript accumulates to a low level because the petD 5Ј processing site has either been compromised (∆P) or deleted (FUD6).…”