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 Delta26 results in reduced accumulation of atpB transcripts and the chloroplast ATPase beta-subunit, leading to weakly photosynthetic growth. Of the residual atpB mRNA in Delta26, 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 beta-subunit which accumulates in Delta26. 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.

FIG. 1

The Chlamydomonas chloroplast atpB region and constructs used in this work. (A) Map of the 7.6-kb BamHI fragment of the Chlamydomonas chloroplast genome. A portion of the large IR of the chloroplast genome is shown as an open arrow. The inverted repeat downstream of the atpB coding region is shown as a stem-loop structure. The extents of the deletions in the chloroplast genomes of strains CC373, Δ26 and Δ26S are indicated by hatched boxes. (B) Detailed view of the sequence of the atpB 3′ UTR in Δ26 and Δ26S. The BglII site into which the rbcL 3′ UTR was inserted in strain R+ is shown in boldface type. The endpoints of the deletion in Δ26 and Δ26S are indicated by triangles.

FIG. 2

(A) Accumulation of total atpB transcripts. A 15-μg amount of total RNA from the indicated strains or yeast tRNA as a control was fixed to nylon filters with a slot blot apparatus, and identical filters were hybridized with ³²P-labeled atpB and psbA probes. (B) Accumulation of the ATPase β-subunit. Total proteins from the indicated strains were fractionated by SDS-polyacrylamide gel electrophoresis, transferred to a nitrocellulose membrane, and incubated sequentially with antibodies directed against the ATPase β-subunit and the D1 protein of photosystem II. Antigenic proteins were visualized by chemiluminescence. (C) Accumulating discrete atpB transcripts. A 15-μg amount of total RNA from the indicated strains was analyzed by filter hybridization with sequential probing for atpB and psbA transcripts. The middle panel is a longer exposure (reproduced from a Fuji imager scan) which reveals the discrete processed transcripts in Δ26. Note that the size of atpB mRNA in the R+ transformant is 2.1 kb, compared to 1.9 kb in wild-type cells (36). WT, wild type.

FIG. 3

Quantification of atpB transcript and ATPase β-subunit protein levels. The total (slot blot) or discrete (gel blot) atpB transcript levels and the ATPase β-subunit levels were calculated from analyzing multiple (at least four) experiments as shown in Fig. 2. The signals were quantified with a Fuji-Imaging analyzer, and in both cases the level of the atpB transcript was normalized to that of the psbA RNA and is presented as a fraction of the amount in wild-type (WT) cells.

FIG. 4

Distribution of atpB transcripts in polysome gradients. Lysates from the strains indicated at the right were sedimented through analytical 15 to 55% sucrose gradients. Ten fractions were collected, and the RNA purified from the lysate of whole cells (T) as well as fractions 2 to 9 was assayed for atpB mRNA by RNA gel blot hybridization. (A) Ethidium bromide staining of the gel for wild-type cells to reveal the distribution of the rRNAs (similar gels were obtained for other strains but are not shown). 23S* is an in vivo breakdown product of plastid 23S rRNA (28). The migrations of DNA molecular weight markers in panel A and the atpB transcript (1.9 kb in the wild type [WT] and 2.1 kb in R+ [36]) are indicated at the left. (F) Gradient fractions derived from Δ26S (the ones shown in panel D) probed with a psbA-specific fragment. The relative exposure times for the panels were as follows: C > D > B = E = F.

FIG. 5

Distribution of atpB transcripts in polysome gradients with or without EDTA. Lysates from Δ26 (A to D) and Δ26S (E and F) were sedimented and analyzed as described in the legend to Fig. 4. EDTA treatment is described in Materials and Methods.

FIG. 6

Quantification of total atpB transcripts in polysome gradient fractions. (A) Distribution of total atpB transcripts in polysome gradient fractions of the indicated strains was analyzed as described in the legend to Fig. 4, except that slot blot rather than gel blot analysis was used. (B) The signals were quantified with a Fuji-Imaging analyzer, and in both cases the level of the atpB transcript in each fraction is presented as a fraction of the total amount of atpB transcript, which was set to 100% (B). Fraction 1 was omitted for the reasons described in the text. WT, wild type.

FIG. 7

Distribution of rbcL mRNA in polysome gradient fractions. Lysates from wild-type cells were fractionated and analyzed as described in the legend to Fig. 4. The probes used in panels A and B are indicated on the map of the rbcL region shown at the bottom. Arrow, the 3′ end of mature rbcL mRNA.