Use of plant chloroplast RNA-binding proteins as orthogonal activators of chloroplast transgenes in the green alga Chlamydomonas reinhardtii

Chloroplast subfractions were tested with a UV cross-linking assay for proteins that bind to the 5′ untranslated region of the chloroplast psbC mRNA of the green alga Chlamydomonas reinhardtii. These analyses revealed that RNA-binding proteins of 30–32, 46, 47, 60, and 80 kD are associated with chloroplast membranes. The buoyant density and the acyl lipid composition of these membranes are compatible with their origin being the inner chloroplast envelope membrane. However, unlike previously characterized inner envelope membranes, these membranes are associated with thylakoids. One of the membrane-associated RNA-binding proteins appears to be RB47, which has been reported to be a specific activator of psbA mRNA translation. These results suggest that translation of chloroplast mRNAs encoding thylakoid proteins occurs at either a subfraction of the chloroplast inner envelope membrane or a previously uncharacterized intra-chloroplast compartment, which is physically associated with thylakoids.

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RNA-binding protein DUS16 plays an essential role in primary miRNA processing in the unicellular alga Chlamydomonas reinhardtii

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1523230113 ◽

2016 ◽

Vol 113 (38) ◽

pp. 10720-10725 ◽

Cited By ~ 10

Author(s):

Tomohito Yamasaki ◽

Masayuki Onishi ◽

Eun-Jeong Kim ◽

Heriberto Cerutti ◽

Takeshi Ohama

Keyword(s):

Chlamydomonas Reinhardtii ◽

Essential Role ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Binding Protein ◽

Rna Binding Protein ◽

Mature Mirnas ◽

Mirna Biogenesis ◽

Dsrna Binding

Canonical microRNAs (miRNAs) are embedded in duplexed stem–loops in long precursor transcripts and are excised by sequential cleavage by DICER nuclease(s). In this miRNA biogenesis pathway, dsRNA-binding proteins play important roles in animals and plants by assisting DICER. However, these RNA-binding proteins are poorly characterized in unicellular organisms. Here we report that a unique RNA-binding protein, Dull slicer-16 (DUS16), plays an essential role in processing of primary-miRNA (pri-miRNA) transcripts in the unicellular green alga Chlamydomonas reinhardtii. In animals and plants, dsRNA-binding proteins involved in miRNA biogenesis harbor two or three dsRNA-binding domains (dsRBDs), whereas DUS16 contains one dsRBD and also an ssRNA-binding domain (RRM). The null mutant of DUS16 showed a drastic reduction in most miRNA species. Production of these miRNAs was complemented by expression of full-length DUS16, but the expression of RRM- or dsRBD-truncated DUS16 did not restore miRNA production. Furthermore, DUS16 is predominantly localized to the nucleus and associated with nascent (unspliced form) pri-miRNAs and the DICER-LIKE 3 protein. These results suggest that DUS16 recognizes pri-miRNA transcripts cotranscriptionally and promotes their processing into mature miRNAs as a component of a microprocessor complex. We propose that DUS16 is an essential factor for miRNA production in Chlamydomonas and, because DUS16 is functionally similar to the dsRNA-binding proteins involved in miRNA biogenesis in animals and land plants, our report provides insight into this mechanism in unicellular eukaryotes.

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Extreme heterogeneity of polyadenylation sites in mRNAs encoding chloroplast RNA-binding proteins in Nicotiana plumbaginifolia

Plant Molecular Biology ◽

10.1007/bf00020402 ◽

1995 ◽

Vol 28 (3) ◽

pp. 569-574 ◽

Cited By ~ 9

Author(s):

Ulrich Klahre ◽

Maja Hemmings-Mieszczak ◽

Witold Filipowicz

Keyword(s):

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Nicotiana Plumbaginifolia ◽

Polyadenylation Sites ◽

Chloroplast Rna

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RNA-stabilization factors in chloroplasts of vascular plants

Essays in Biochemistry ◽

10.1042/ebc20170061 ◽

2018 ◽

Vol 62 (1) ◽

pp. 51-64 ◽

Cited By ~ 18

Author(s):

Nikolay Manavski ◽

Lisa-Marie Schmid ◽

Jörg Meurer

Keyword(s):

Stress Responses ◽

Vascular Plants ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Rna Degradation ◽

Decay Rates ◽

Transcriptional Level ◽

Chloroplast Gene Expression ◽

Chloroplast Rna

In contrast to the cyanobacterial ancestor, chloroplast gene expression is predominantly governed on the post-transcriptional level such as modifications of the RNA sequence, decay rates, exo- and endonucleolytic processing as well as translational events. The concerted function of numerous chloroplast RNA-binding proteins plays a fundamental and often essential role in all these processes but our understanding of their impact in regulation of RNA degradation is only at the beginning. Moreover, metabolic processes and post-translational modifications are thought to affect the function of RNA protectors. These protectors contain a variety of different RNA-recognition motifs, which often appear as multiple repeats. They are required for normal plant growth and development as well as diverse stress responses and acclimation processes. Interestingly, most of the protectors are plant specific which reflects a fast-evolving RNA metabolism in chloroplasts congruent with the diverging RNA targets. Here, we mainly focused on the characteristics of known chloroplast RNA-binding proteins that protect exonuclease-sensitive sites in chloroplasts of vascular plants.

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