Abstract
The post-transcriptional addition of uridines to the 3'-end of RNAs is an important regulatory process that is critical for coding and noncoding RNA stability. In fission yeast and metazoans this untemplated 3'-uridylylation is catalysed by a single family of terminal uridylyltransferases (TUTs) whose members are adapted to specific RNA targets. In Schizosaccharomyces pombe the TUT Cid1 is responsible for the uridylylation of polyadenylated mRNAs, targeting them for destruction. In metazoans, the Cid1 orthologues ZCCHC6 and ZCCHC11 uridylate histone mRNAs, targeting them for degradation, but also uridylate microRNAs, altering their maturation. Cid1 has been studied as a model TUT that has provided insights into the larger and more complex metazoan enzyme system. In this paper, two strategies are described that led to improvements both in the crystallogenesis of Cid1 and in the resolution of diffraction by ∼1.5 Å. These advances have allowed high-resolution crystallographic studies of this TUT system to be initiated.
Highlights
The addition of untemplated nucleotides to the 30-ends of RNAs is a critical regulatory mechanism involved in a host of cellular and physiological processes including cell-cycle control, cell proliferation and differentiation, and embryonic development (Norbury, 2013; Yates et al, 2013)
The enzymes responsible for RNA uridylylation are the terminal RNA uridylyltransferases (TUTs), which belong to a conserved family of nucleotidyltransferases and are related to DNA polymerase and poly(A) polymerase (Rissland et al, 2007; Yates et al, 2012)
A pGEX6P-1 truncated Cid1 construct was generated as described in Rissland et al (2007), and the K133A/ R137A/R277A/K282A mutant was generated by successive site-directed mutagenesis procedures as described in Yates et al (2012) using the primers described in Table 1, where the suffixes ‘F’ and ‘R’ signify forward and reverse primers, respectively, and underlined capitalized nucleotides denote mutagenized codons
Summary
The addition of untemplated nucleotides to the 30-ends of RNAs is a critical regulatory mechanism involved in a host of cellular and physiological processes including cell-cycle control, cell proliferation and differentiation, and embryonic development (Norbury, 2013; Yates et al, 2013). The enzymes responsible for RNA uridylylation are the terminal RNA uridylyltransferases (TUTs), which belong to a conserved family of nucleotidyltransferases and are related to DNA polymerase and poly(A) polymerase (Rissland et al, 2007; Yates et al, 2012). Several structures of Cid have been solved as an apo enzyme and bound to UTP and substrate mimics (Lunde et al, 2012; Yates et al, 2012; Munoz-Tello et al, 2012, 2014). We present strategies to improve the crystallization and diffraction resolution of Cid for further high-resolution crystallographic studies
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