Identification and Characterization of Novel miRNAs in Chlamydomonas reinhardtii by Computational Methods.

BACKGROUND: MicroRNAs (miRNAs) are endogenous small non-coding RNAs with 18-24 nucleotides in length, which have important roles in posttranscriptional gene regulation. The resemblance of miRNA biogenesis in unicellular green algae and those in plants suggests probable evolutionary conserved pathways. This conservation provides a ground towards prediction of new homologs via computational biology. METHODS: Here, conserved miRNA genes in Chlamydomonas reinhardtii and plants were examined through homology alignment. Previously known and unique plant miRNAs were BLASTed against expressed sequence tags (ESTs) and genomic survey sequences (GSSs) of C. reinhardtii. All candidate sequences with appropriate fold back structures were screened according to a series of miRNA filtering criteria. RESULTS: Homologous miRNAs (17), belonging to 9 miRNA gene families were predicted. Interestingly and for the first time, a miRNA family of genes was localized to chloroplast. Again and for the first time, here we report identification of C. reinhardtii miRNA orthologs in plants and animals. miRNA target genes were identified based on their sequence complementarities to the respective miRNAs using psRNATarget against C. reinhardtii, Unigene, and DFCI Gene Index (CHRGI). Totally, 152 potential target sites were identified. From the predicted miRNAs, 7 miRNAs had no target sequence in C. reinhardtii protein coding genes. CONCLUSION: Identifying miRNA and their target transcript(s) would be useful for other research concerned with the function and regulatory mechanisms of C. reinhardtii miRNAs and helps researchers to better understand the nature of its extensive metabolic flexibility and environmental compatibility to survive in distinct environmental niches and nutrient availability.

Rapid evolution of RNA editing sites in a small non-essential plastid gene.

Chloroplast RNA editing proceeds by C-to-U transitions at highly specific sites. Here, we provide a phylogenetic analysis of RNA editing in a small plastid gene, petL, encoding subunit VI of the cytochrome b6f complex. Analyzing representatives from most major groups of seed plants, we find an unexpectedly high frequency and dynamics of RNA editing. High-frequency editing has previously been observed in plastid ndh genes, which are remarkable in that their mutational inactivation does not produce an obvious mutant phenotype. In order to test the idea that reduced functional constraints allow for more flexible evolution of RNA editing sites, we have created petL knockout plants by tobacco chloroplast transformation. We find that, in the higher plant tobacco, targeted inactivation of petL does not impair plant growth under a variety of conditions markedly contrasting the important role of petL in photosynthesis in the green alga Chlamydomonas reinhardtii. Together with a low number of editing sites in plastid genes that are essential to gene expression and photosynthetic activity, these data suggest that RNA editing sites may evolve more readily in those genes whose transitory loss of function can be tolerated. Accumulated evidence for this 'relative neutrality hypothesis for the evolution of plastid editing sites' is discussed.

Polyadenylation of three classes of chloroplast RNA in Chlamydomonas reinhadtii.

Three classes of RNA, represented by atpB and petD mRNAs, Arg and Glu tRNAs, and 5S rRNA, were found to exist in polyadenylated form in Chlamydomonas reinhardtii chloroplasts. Sequence analysis of cDNA clones derived from reverse transcriptase-polymerase chain reaction protocols used to select polyadenylated RNAs revealed that, at least for the mRNAs and tRNAs, there are three apparent types of polyadenylation. In the first case, the poly(A) tail is added at or near the mature 3' end, even when this follows a strong secondary structure. In the second case, the tail is added to pre-mRNA or pre-tRNA, suggesting a possible competition between polyadenylation and RNA-processing pathways. Finally, in all cases, the poly(A) tail can be added internally, possibly as a part of an RNA-decay pathway. The tails found in Chlamydomonas chloroplasts differ from those of spinach chloroplasts in adenine content, being nearly homopolymeric (>98% adenine) versus 70% in spinach, and are similar in length to those of Escherichia coli, being mostly between 20 and 50 nt. In vitro assays using a Chlamydomonas chloroplast protein extract showed that a 3' end A25 tail was sufficient to stimulate rapid degradation of atpB RNA in vitro, with a lesser effect for petD, and only minor effects on trnE. We therefore propose that polyadenylation contributes to mRNA degradation in Chlamydomonas chloroplasts, but that its effect may vary.