Robert G. Drager

The sequence and structure of the 3′-untranslated regions of chloroplast transcripts are important determinants of mRNA accumulation and stability

A general characteristic of the 3′-untranslated regions (3′ UTRs) of plastid mRNAs is an inverted repeat (IR) sequence that can fold into a stem-loop structure. These stem-loops are RNA 3′-end processing signals and determinants of mRNA stability, not transcription terminators. Incubation of synthetic RNAs corresponding to the 3′ UTRs of Chlamydomonas chloroplast genes atpB and petD with a chloroplast protein extract resulted in the accumulation of stable processing products. Synthetic RNAs of the petA 3′ UTR and the antisense strand of atpB 3′ UTR were degraded in the extract. To examine 3′ UTR function in vivo, the atpB 3′ UTR was replaced with the 3′ UTR sequences of the Chlamydomonas chloroplast genes petD, petD plus trnR, rbcL, petA and E. coli thrA by biolistic transformation of Chlamydomonas chloroplasts. Each 3′ UTR was inserted in both the sense and antisense orientations. The accumulation of both total atpB mRNA and ATPase β-subunit protein in all transformants was increased compared to a strain in which the atpB 3′ UTR had been deleted. However, the level of discrete atpB transcripts in transformants containing the antisense 3′ UTR sequences was reduced to approximately one-half that of transformants containing the 3′ UTRs in the sense orientation. These results imply that both the nucleotide sequences and the stem-loop structures of the 3′ UTRs are important for transcript 3′-end processing, and for accumulation of the mature mRNAs.

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 theChlamydomonas chloroplast atpB 3′ IR in strain Δ26 results in reduced accumulation of atpB transcripts and the chloroplast ATPase β-subunit, leading to weakly photosynthetic growth. Of the residualatpB mRNA in Δ26, 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 atpBtranscripts 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 β-subunit which accumulates in Δ26. Analysis of chloroplast rbcLmRNA 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.

The 3′ untranslated regions of chloroplast genes inChlamydomonas reinhardtii do not serve as efficient transcriptional terminators

A general characteristic of the 3′ untranslated regions of plastid mRNAs is an inverted repeat sequence that can fold into a stem-loop structure. These stem-loops are superficially similar to structures involved in prokaryotic transcription termination, but were found instead to serve as RNA 3′ end processing signals in spinach chloroplasts, and in theatpB mRNA ofChlamydomonas reinhardtii chloroplasts. In order to carry out a broad study of the efficiency of the untranslated sequences at the 3′ ends of chloroplast genes inChlamydomonas to function as transcription terminators, we performed in vivo run-on transcription experiments usingChlamydomonas chloroplast transformants in which different 3′ ends were inserted into the chloroplast genome between apetD promoter and a reporter gene. The results showed that none of the 3′ ends that were tested, in either sense or antisense orientation, prevented readthrough transcription, and thus were not highly efficient transcription terminators. Therefore, we suggest that most or all of the 3′ ends of mature mRNAs inChlamydomonas chloroplasts are formed by 3′ end processing of longer precursors.

A nucleus-encoded suppressor defines a new factor which can promote petD mRNA stability in the chloroplast of Chlamydomonas reinhardtii

Abstract Mutations in the Chlamydomonas reinhardtii nuclear gene MCD1 specifically destabilize the chloroplast petD mRNA, which encodes subunit IV of the cytochrome b6/f complex. The MCD1 gene product is thought to interact with the mRNA 5′ end to protect it from degradation by a 5′ → 3′ exoribonuclease and may also have a role in translation initiation. Here we report the isolation and characterization of a semidominant, allele-specific, nucleus-encoded suppressor of the mcd1-2 mutation. The suppressor mutation, which defines a new locus MCD2, allows accumulation of 10% of the wild-type level of petD mRNA and as much as 50% of the wild-type subunit IV level. Taken together, these results suggest the suppressor mutation restores photosynthetic growth by stabilizing petD mRNA. In addition, it may promote increased translational efficiency, an inference supported by direct measurements of the subunit IV synthesis rate. Thus, both MCD1 and MCD2 may participate in both chloroplast RNA stability and translation initiation.

Chloroplast RNA Synthesis and Processing

Transcription and RNA maturation are two essential steps in gene expression. In chloroplasts, transcription is carried out by at least two biochemically and genetically separable activities, which may participate in establishing different basal expression rates for ribosomal RNAs, transfer RNAs and protein-coding genes. Because chloroplast RNA polymerases do not generally terminate transcription at sites corresponding to the 3′ termini of mature transcripts, these termini must be formed by RNA processing events. In Chlamydomonas reinhardtii chloroplasts, it appears that most or all transcript 5′-ends are also formed by RNA processing rather than by transcription initiation. Thus, RNA processing converts primary transcripts of generally unknown dimensions to the mature, accumulating transcripts. Molecular, genetic and biochemical approaches have been used to unravel the chloroplast transcription and RNA processing machinery, with the most information gained to date from the analysis of chimeric reporter genes introduced into chloroplasts by biolistic transformation. The picture painted bythese data reveals both similarities and differences between these processes in Chlamydomonas and land plants. However, some perceived differences, particularly based on the phenotypes of nuclear mutants which affect chloroplast mRNA metabolism, may reflect selection or screening procedures and thus may mask an overall congruity between gene expression mechanisms in the chloroplasts of all organisms.