A Deadenylase Assay by Size-Exclusion Chromatography

The shortening of the 3′-end poly(A) tail, also called deadenylation, is crucial to the regulation of mRNA processing, transportation, translation and degradation. The deadenylation process is achieved by deadenylases, which specifically catalyze the removal of the poly(A) tail at the 3′-end of eukaryotic mRNAs and release 5′-AMP as the product. To achieve their physiological functions, all deadenylases have numerous binding partners that may regulate their catalytic properties or recruit them into various protein complexes. To study the effects of various partners, it is important to develop new deadenylase assay that can be applied either in vivo or in vitro. In this research, we developed the deadenylase assay by the size-exclusion chromatography (SEC) method. The SEC analysis indicated that the poly(A) or oligo(A) substrate and the product AMP could be successfully separated and quantified. The enzymatic parameters of deadenylase could be obtained by quantifying the AMP generation. When using the commercial poly(A) as the substrate, a biphasic catalytic process was observed, which might correlate to the two distinct states of poly(A) in the commercial samples. Different lots of commercial poly(A) had dissimilar size distributions and were dissimilar in response to the degradation of deadenylase. The deadenylation pattern, processive or distributive, could also be investigated using the SEC assay by monitoring the status of the substrate and the generation kinetics of AMP and A2. The SEC assay was applicable to both simple samples using the purified enzyme and complex enzyme reaction conditions such as using protein mixtures or crude cell extracts as samples. The influence of solutes with absorption at 254 nm could be successfully eliminated by constructing the different SEC profiles

mRNA cap binding proteins: effects on abscisic acid signal transduction, mRNA processing, and microarray analyses.

International audienceThe plant hormone abscisic acid (ABA) intricately regulates a multitude of processes during plant growth and development. Recent studies have established a connection between genes participating in various steps of cellular RNA metabolism and the ABA signal transduction machinery. In this chapter we focus on the plant nuclear mRNA cap binding proteins, CBP20 and CBP80. We summarize and report recent findings on their effects on cellular signal transduction networks and mRNA processing events. ABA hypersensitive 1 (abh1) harbors a gene disruption in the Arabidopsis CBP80 gene. Loss-of-function mutation of ABH1 can also result in an early flowering phenotype in the Arabidopsis accession C24. abh1 revealed noncoding cis-natural antisense transcripts (cis-NATs) at the CONSTANS locus in wild-type plants with elevated cis-NAT expression in the mutant. abh1 also revealed an influence on the splicing of the MADS box transcription factor Flowering Locus C pre-mRNA, which may result in the regulation of flowering time. Furthermore, new experiments analyzing complementation of cpb20 with site-directed cpb20 mutants provide evidence that the CAP binding activity of CBP20 is essential for the observed cbp-associated phenotypes. In conclusion, mutants in genes participating in RNA processing provide excellent tools to uncover novel molecular mechanisms for the regulation of RNA metabolism and of signal transduction networks in wild-type plants