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

Abstract

In Chlamydomonas chloroplasts, atpB pre-mRNA matures through a two-step process. Initially, endonuclease cleavage occurs 8 -10 nt downstream of the mature 3 end, which itself lies at the end of a stem-loop-forming inverted repeat (IR) sequence. This intermediate product is then trimmed by a 3 3 5 exonuclease activity. Although the initial endonucleolytic cleavage by definition generates two products, the downstream product of atpB pre-mRNA endonucleolytic processing cannot be detected, even transiently. This product thus appears to be highly unstable, and it can be hypothesized that specific mechanisms exist to prevent its accumulation. In experiments described here, the atpB 3 maturation site was placed upstream of reporter genes in vivo. Constructs containing both the IR and endonuclease cleavage site (ECS) did not accumulate the reporter gene mRNA, whereas constructs containing only the IR did accumulate the reporter mRNA. The ECS alone gave an intermediate result, suggesting that the IR and ECS act synergistically. Additional secondary structures were used to test whether 5 3 3 and/or 3 3 5 exonuclease activities mediated degradation. Because these structures did not prevent degradation, rapid endonucleolytic cleavages most likely trigger RNA destruction after ECS cleavage. On the other hand, fragments resulting from cleavage within the endogenous atpB mRNA could occasionally be detected as antisense transcripts of the adjacent reporter genes. Because endonuclease cleavages are also involved in the 5 maturation of chloroplast mRNAs, where only the downstream cleavage product accumulates, it appears that chloroplast endoribonuclease activities have evolved mechanisms to selectively stabilize different ECS products.Maturation of mRNA can involve multiple steps in both prokaryotic and eukaryotic systems, and results in functional transcripts with a stability and subcellular localization appropriate to their functions. RNA sequence and secondary structure, along with ribonucleases and a variety of accessory factors, are used to achieve these goals. Built into this process is the necessity to recognize nonfunctional RNAs and eliminate them; destruction of nonsense codon-containing mRNAs is a good example of the complexity of such surveillance mechanisms (reviewed in Ref. 1).Our laboratory has focused on 5Ј end and 3Ј end maturation of chloroplast mRNAs, using both vascular plants and the unicellular green alga Chlamydomonas reinhardtii as models. In this respect, chloroplast transcripts have mostly prokaryotic features such as the lack of a trimethylguanosine 5Ј cap, a 3Ј stem-loop-forming inverted repeat (IR) 1 structure, and they are destabilized by polyadenylation (reviewed in Refs. 2 and 3). Processing of chloroplast mRNAs, with rare exceptions, depends on nucleus-encoded proteins, several of which have been identified genetically through screens for plants or Chlamydomonas strains unable to carry out photosynthesis. The phenotypes suggest that defects in intercistronic processing of polycistronic transcripts...

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

“…In fact, the atpB transcripts in ⌬19 and ⌬21 seemed to be processed correctly, consistent with competition between RNA processing and decay pathways. Indeed, cleavage at the atpB 3Ј-UTR ECS is accompanied by rapid degradation of downstream sequences (24,33), which in this case would be the poly(A) moiety. Processing can also influence degradation in other systems.…”

Antisense Transcript and RNA Processing Alterations Suppress Instability of Polyadenylated mRNA in Chlamydomonas Chloroplasts

“…Although many such RNAs may be synthesized as a result of the compact nature of chloroplast genomes, because of the lack of efficient termination, they are likely to be unstable. For example, Chlamydomonas chloroplasts have a highly efficient mechanism for degrading atpB mRNA downstream of its eventual maturation site (Hicks et al, 2002), a phenomenon that might in fact help the chloroplast avoid the accumulation of dsRNA, whose stability might be difficult to control. Furthermore, the Chlamydomonas chloroplast genome is atypically asymmetric (i.e., although genes are encoded on both strands, they are mostly in blocks that are likely to be independently transcribed).…”

Chloroplast RNase J compensates for inefficient transcription termination by removal of antisense RNA

“…Thus, primary triphosphorylated transcripts are thought to be largely impervious to RNase J exonucleolytic attack. Chloroplasts are known to have a net 59 / 39 RNA degradation pathway (Drager et al 1999;Hicks et al 2002), and RNase J has also been proposed to catalyze 59 end trimming of monocistronic RNAs and polycistronic transcript derivatives, being stalled by gene-specific RNA binding proteins (see Fig. 2 in Barkan 2011).…”