Gene elements that affect the longevity of rbcL sequence-containing transcripts in Chlamydomonas reinhardtii chloroplasts

Abstract

The chloroplast gene rbcL encodes the large subunit of the CO2-fixing enzyme ribulose-bisphosphate carboxylase. In previous work a target for photo-accelerated degradation of Chlamydomonas reinhardtii rbcL transcripts in vivo was found to lie within the first 63 nucleotides, and a sequence element required for increasing the longevity of transcripts of rbcL-reporter genes was found to occur between nucleotides 170 and 350. Photo-accelerated degradation of rbcL transcripts has been found to require nucleotides 21 to 41. Transcript nucleotides lying between 329 and 334 and between 14 and 27 are essential for stabilizing transcripts in vivo; mutations in either region reduce the longevity of transcripts. It is postulated that the effectiveness of photo-accelerated endonuclease attacks on the nucleotide 21 to 41 region is reduced by physical blockage or distortion of the target sequence by interacting proteins that associate with nucleotides in the 14 to 27 and 329 to 334 regions of the transcripts. Both the nucleotide ؉329 to ؉334 stabilizing sequence of rbcL and a transcription enhancing sequence that lies between ؉126 and ؉170 encode well conserved (cyanobacteria through angiosperms) amino acid sequences; the evolution of expression control elements within the protein coding sequence of rbcL is considered.

Chloroplast Biogenesis of Photosystem II Cores Involves a Series of Assembly-Controlled Steps That Regulate Translation

“…Only when at least 60 nucleotides of the psbA coding sequence were added in frame, upstream of the reporter gene, did the chimeric mRNA accumulate, enabling the phototrophic growth of the transformants (data not shown). These results point to the existence of cis-acting element stabilizing psbA-driven transcripts within the coding sequence of psbA, as already described for other chloroplast genes in C. reinhardtii (Singh et al, 2001) or for psbA in cyanobacteria (Kulkarni and Golden, 1997). Thus, we used for the rest of our study transformed strains expressing cytochrome f translated under the control of the psbA 59UTR, followed by the 60 first nucleotides of the psbA coding sequence, hereafter called bAf because they express a 59psbA-driven cytochrome f.…”

Specific sequence elements in the 5′ untranslated regions of rbcL and atpB gene mRNAs stabilize transcripts in the chloroplast of Chlamydomonas reinhardtii

“…Rates of transcription of chimeric rbc L 59 end:GUS and atpB 59 end:GUS genes relative to the endogenous Chlamydomonas chloroplast genes rbc L and atpB+ Phosphate-starved cells were allowed to incorporate [ 32 P]-orthophosphate for 10 min and 20 min in the dark+ Total RNA was isolated and hybridized for 72 h to probes specific for the GUS, atpB, rbc L, and pUC18 sequences (see Materials and Methods) spotted on a nylon membrane in a slot-blot apparatus (Hoefer Scientific Instruments, San Francisco, California)+ Signals were visualized by exposure of the membranes to X-ray film for approximately 48 h and quantified using Kodak 1D image analysis software+ Numbers above the autoradiograms denote the positions of mutated nucleotides in the rbc L or atpB 59 UTRs (see Fig+ 1)+ Ratios given to the right of the autoradiograms were calculated relative to the rates of phosphate incorporation into transcripts of the endogenous rbc L (upper panel) and atpB (lower panel) genes (set to 1+00) that serve as internal standards+ Relative rates of transcription of rbc L 59 end:GUS genes are only 20 to 30% of the rate of transcription of the endogenous rbc L gene because the rbc L 59 ends fused to the GUS coding region lack a transcriptional enhancer that is located in the coding region of the endogenous rbc L gene (Klein et al+, 1994)+ Separate sequence elements in the rbc L 59 UTR mediate longevity and light /dark stability of transcripts It has been reported (Salvador et al+, 1993b) that transcripts of chimeric rbc L 59 end:GUS genes containing the rbc L promoter and 59 UTR are rapidly degraded in Chlamydomonas chloroplast transformants upon illumination of the cells+ The light-induced decay was shown to be conferred to rbc L 59 UTR:GUS transcripts by the rbc L 59 UTR sequence (Salvador et al+, 1993b)+ The in vivo mutational analyses of the rbc L 59 UTR described above were done in cultures of Chlamydomonas grown in 12-h light/12-h dark cycles and cells for isolation of total RNA and for determination of rates of transcription were collected at the end of the dark period (see Materials and Methods)+ It seemed possible that the rbc L 59 UTR sequence element (positions ϩ38 to ϩ47; Fig+ 1) found in this study to be required for stability of chimeric rbc L 59 UTR:GUS transcripts is also involved in light/dark regulation of transcript stability+ To test this notion, GUS transcript levels were determined in the dark and in the light in chloroplast transformants harboring mutated rbc L 59 end:GUS genes (mutations at positions ϩ39, ϩ40, ϩ42, ϩ45, and ϩ46 in the rbc L 59 UTR; Fig+ 4)+ GUS transcripts that were already destabilized by a mutation in the sequence element of the rbc L 59 UTR and that accumulated in the dark to very low levels in Chlamydomonas chloroplast transformants (Fig+ 2), still showed the typical light/dark regulation of abundance (Fig+ 4)+ This suggests that the light/dark-regulated mechanism of transcript (de-) stabilization is distinct from the RNA decay mechanism functioning in turnover of rbc L chloroplast transcripts in the dark, the former probably involving sequences in the rbc L 59 UTR that are different from the element defined in this study (Singh et al+, 2001)+…”

“…The finding that altering the sequence element in the rbc L 59 UTR influences the longevity of transcripts in the chloroplast of Chlamydomonas but does not affect the light/dark regulation of transcript stability (Fig+ 4) points to a light/dark-regulated pathway of RNA breakdown that differs from the general pathway of mRNA turnover in the chloroplast+ The light/dark-regulated pathway has been shown to be linked to the redox state in the chloroplast (Salvador & Klein, 1999) and probably involves chloroplast proteins that function as redox carriers+ The recent delineation in the rbc L 59 UTR of cis-acting sequences at positions ϩ21 to ϩ41 that play a role in light/dark regulation of rbc L transcript stability (Singh et al+, 2001) should aid in identifying the molecular components and mechanisms involved in control of transcript longevities in chloroplasts+…”

The stem-loop region of the tobacco psbA 5′UTR is an important determinant of mRNA stability and translation efficiency

“…In contrast, the 5¢UTRs appear to be of crucial importance for both mRNA stability and translation efficiency, as indicated by various analyses of chimeric transcripts and nuclear mutants (Mayfield et al 1994;Higgs et al 1999;Nickelsen et al 1999;Vaistij et al 2000;Singh et al 2001). The cis -acting expression determinants have been described for a number of chloroplast mRNAs.…”

“…Some of these elements contain sequences that have the potential to form secondary structures. For instance, the loop sequence and several stem bases of the 5¢ end stem-loop region of the Chlamydomonas rbcL 5¢UTR determine transcript lifetime (Anthonisen et al 2001;Singh et al 2001), and a structural segment at the 5¢ end of the tobacco rbcL 5¢UTR is thought to promote mRNA stability, thus compensating for the lower rate of transcription of this mRNA in the dark (Shiina et al 1998). In Chlamydomonas, the putative stem-loop region of the psbA 5¢UTR (Mayfield et al 1994), the structural element III of the petD 5¢UTR (Higgs et al 1999), the secondary structures embedded in the 100-nt central region of the psbC 5¢UTR (Rochaix et al 1989;Zerges et al 1997) and the 3¢ half of the 266-nt rps75¢UTR (Fargo et al 1999) appear to be essential for translation initiation.…”