Biochemical and biophysical characterization of the deadenylase CrCaf1 from Chlamydomonas reinhardtii.
The modulation of mRNA turnover has been increasingly recognized as a hotpoint for gene expression regulation at the post-transcriptional level. In eukaryotic cells, most mRNAs are degraded via the deadenylation-dependent pathway, in which the removal of the poly(A) tail is the initial and rate-limiting step. Caf1, a deadenylase specifically degrades poly(A) from the 3'-end, is highly conserved from yeast to mammalians. Caf1s in higher plants have been shown to be involved in plant development and stress response. However, little is known about the biochemical and biophysical properties of Caf1s in plants. In this research, we cloned the crcaf1 gene from Chlamydomonas reinhardtii and studied the properties of the recombinant proteins. The results showed that CrCaf1 was a deadenylase with conserved sequence motifs, structural features, and catalytic properties of the Caf1 family. CrCaf1 degraded poly(A) in a distributive mode with the optimal reacting conditions at pH 7 and 35°C. CrCaf1 had similar activity when coordinated with Mg(2+) and Mn(2+), while the enzyme bound to Ca(2+) or Zn(2+) was almost inactivated. Zn(2+) could induce CrCaf1 aggregation with the disruption of the native structure, while Mg(2+), Mn(2+) and Ca(2+) could stabilize CrCaf1 against thermal denaturation by reducing protein aggregation. Among the various metal ions, Mn(2+) showed the strongest protective effect on CrCaf1 stability, implying that Mn(2+) might play a role in regulating CrCaf1 stability in the C. reinhardtii cells under some stressed conditions. These findings provide a starting point for further investigation of the physiological functions of CrCaf1 in C. reinhardtii.
State Key Laboratory of Biomembrane and Membrane Biotechnology, School of Life Sciences, Tsinghua University, Beijing, China.
Jia-Quan
JQ
Zhang
Guang-Jun
GJ
He
Yong-Bin
YB
Yan
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D055592:Biophysical Phenomena / Q000187:drug effects; D002413:Cations, Divalent / Q000494:pharmacology; D016825:Chlamydomonas reinhardtii / Q000187:drug effects / Q000201:enzymology*; D018360:Crystallography, X-Ray; D004795:Enzyme Stability / Q000187:drug effects; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry; D012260:Ribonucleases / Q000737:chemistry / Q000378:metabolism*; D016415:Sequence Alignment; D013696:Temperature
D002413:Cations, Divalent; D010940:Plant Proteins; D011994:Recombinant Proteins; D012260:Ribonucleases