Small RNA profiling in Chlamydomonas: insights into chloroplast RNA metabolism

Abstract

In Chlamydomonas reinhardtii, regulation of chloroplast gene expression is mainly post-transcriptional. It requires nucleus-encoded trans-acting protein factors for maturation/stabilization (M factors) or translation (T factors) of specific target mRNAs. We used long- and small-RNA sequencing to generate a detailed map of the transcriptome. Clusters of sRNAs marked the 5′ end of all mature mRNAs. Their absence in M-factor mutants reflects the protection of transcript 5′ end by the cognate factor. Enzymatic removal of 5′-triphosphates allowed identifying those cosRNA that mark a transcription start site. We detected another class of sRNAs derived from low abundance transcripts, antisense to mRNAs. The formation of antisense sRNAs required the presence of the complementary mRNA and was stimulated when translation was inhibited by chloramphenicol or lincomycin. We propose that they derive from degradation of double-stranded RNAs generated by pairing of antisense and sense transcripts, a process normally hindered by the traveling of the ribosomes. In addition, chloramphenicol treatment, by freezing ribosomes on the mRNA, caused the accumulation of 32–34 nt ribosome-protected fragments. Using this ‘in vivo ribosome footprinting’, we identified the function and molecular target of two candidate trans-acting factors.

Systematic sequencing of chloroplast transcript termini fromArabidopsis thalianareveals >200 transcription initiation sites and the extensive imprints of RNA-binding proteins and secondary structures

“…The processed petL 5' end is easily identifiable, however (see below). This suggests rapid maturation of the primary transcript, a phenomenon that is more prevalent in Chlamydomonas reinhardtii chloroplasts, where a recent transcriptome analysis revealed only 23 TSS, albeit using an entirely different method (48). Terminome-Seq also confirmed our earlier discovery of TSS upstream of trnC, trnF and trnN (22).…”

A key role for phosphorylation of PsbH in the biogenesis and repair of photosystem II in Chlamydomonas

“…pAR14- psbC / aadA was obtained by insertion of a recyclable aadA selection cassette (as a MluI - MluI fragment) into a plasmid containing a 3.7 kb XbaI-SalI psbC fragment, at an engineered MluI restriction site 733 bp upstream of the GTG start codon of psbC . This site turned out to be located in the 3’UTR of the ycf2 gene (Cavaiuolo et al 2017), but after excision of the aadA marker the control transformants with the residual footprint ( C T ) had no apparent phenotype.…”

The accumulation of chloroplast small RNAs in unicellular algaChlamydomonas reinhardtiiis affected by nitrogen deprivation

“…The locations of each N-responsive csRNAs on the chloroplast genome were investigated and stated in Tables 2,3 and 4. The assignment of the positions in terms of 5’ and 3’ untranslated regions (UTR) of chloroplast protein-coding genes was based on Cavaiuolo et al . 2017.…”

“…Molecules of ncRNAs were shown to be present not only in the nuclear-cytosolic compartment but also in the chloroplasts of Chlamydomonas reinhardtii (Goldshmidt-Clermont et al . 1991; Cavaiuolo et al . 2017), Arabidopsis thaliana (Lung et al .…”