Novel interaction of two clock‐relevant RNA‐binding proteins C3 and XRN1 in Chlamydomonas reinhardtii

Abstract

Edited by Gianni Cesareni Keywords:Circadian clock RNA metabolism Chlamydomonas reinhardtii a b s t r a c tThe RNA-binding protein CHLAMY1 of the green alga Chlamydomonas reinhardtii consists of two subunits, named C1 and C3 that maintain the period and phase of the circadian clock. Here, we investigated if any of its subunits interact with other clock components involved in RNA metabolism. We found that C3, but not C1 strongly interacts with exoribonuclease XRN1 whose knockout results in low amplitude rhythms. XRN1 is subject to degradation by the proteasome pathway. Its level increases in cells grown at lower ambient temperature simulating night, which was also observed for C3. Our data indicate a network of clock-relevant RNA-binding proteins. Structured summary of protein interactions:XRN1 physically interacts with C3 by anti bait coimmunoprecipitation (View interaction) XRN1 physically interacts with C3 by two hybrid (View interaction) C1 physically interacts with C3 by two hybrid (View interaction)

A Musashi Splice Variant and Its Interaction Partners Influence Temperature Acclimation in Chlamydomonas

“…Recently, we identified the interaction of two C. reinhardtii proteins that integrate temperature information: the clock-relevant RNA-binding protein C3 and XRN1 (Dathe et al, 2012). In C. reinhardtii, knowledge about XRN1 and the members of the downstream signaling cascade is limited.…”

“…We performed a coimmunoprecipitation assay using purified anti-XRN1 antibodies. Proteins of a crude extract from wild-type cells and the xrn1 knockout mutant (Dathe et al, 2012;negative control) were precipitated, separated by SDS-PAGE, sliced into gel pieces covering the range of 17 to ∼260 kD, in-gel digested with trypsin, and subjected to liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) analyses. The experiment was replicated twice.…”

“…To verify the interaction between the 60-kD Musashi variant 12 and XRN1 or C3, we performed a yeast two-hybrid analysis. The mated yeasts carrying 60-kD The interaction of C3 with XRN1 was formerly verified by immunoblotting with anti-C3 antibody and yeast twohybrid analysis (Dathe et al, 2012). Musashi variant 12, XRN1, and C3 in the vectors expressing GAL4-AD or GAL4-BD were used as negative controls ( Fig.…”

“…The effect is stronger during daytime (LD4) compared with early night (LD14). We also investigated XRN1, which has been analyzed previously under these conditions in the presence of a proteasome inhibitor (Dathe et al, 2012). Here, we omitted the inhibitor and concentrated primarily on the full-size protein of ∼198 kD (predicted size) in addition to one prominent degradation product of ∼140 kD.…”

“…The C3 subunit, which bears three RNA recognition motifs (RRMs), is up-regulated at the protein level in cells grown at 18°C. Moreover, a clock-relevant interaction partner of C3, the 5ʹ-3ʹ exoribonuclease XRN1 (also known as Rhythm of Chloroplast86; Matsuo et al, 2008) that is subjected to degradation by the proteasome pathway, is up-regulated at the protein level in cells grown at 18°C in the presence of an inhibitor of the proteasome pathway (Dathe et al, 2012). Therefore, some of the algal clock components that are known currently (Noordally and Millar, 2015;Ryo et al, 2016) also are potential candidates for temperature control.…”