Crystal structure of Arabidopsis terminal uridylyl transferase URT1

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Highlights

  • The URT1 structure we determined in this research represents the first structure of 3′ terminal uridylyl transferase of RNAs from plants.

  • Arabidopsis URT1 as a uridylyl transferase is structurally and functionally similar to Schizosaccharomyces pombe Cid1.

  • URT1 structure determination paves the path towards the illustration of the molecular mechanism of URT1/HESO1 dual TUTases in plants.

Abstract

3′ uridylation is an essential modification associated with coding and noncoding RNA degradation in eukaryotes. In Arabidopsis, HESO1 was first identified as the major nucleotidyl transferase that uridylates most unmethylated miRNAs, and URT1 was later reported to play a redundant but important role in miRNA uridylation when HESO1 is absent. Two enzymes work sequentially and collaboratively to tail different forms of the same miRNAs in vivo. For mRNA, however, URT1 becomes the main enzyme to uridylate the majority of mRNA and repairs their deadenylated ends to restore the binding site for Poly(A) Binding Protein (PABP). HESO1, on the other hand, targets mostly the mRNAs with very short oligo(A) tails and fails in fulfilling the same task. To understand the structural basis these two functional homologues possess for their different substrate preferences and catalytic behaviors, we first determined the crystal structures of URT1 in the absence and presence of UTP. Our structures, together with functional assay and sequence analysis, indicated that URT1 has a conserved UTP-recognition mechanism analogue to the terminal uridylyl transferases from other species whereas HESO1 may evolve separately to recognize UTP in a different way. Moreover, URT1 N552 may be an important residue in interacting with 3′ nucleotide of RNA substrate. The URT1 structure we determined represents the first structure of uridylyl transferase from plants, shedding light on the mechanisms of URT1/HESO1-dependent RNA metabolism.

Introduction

Non-templated uridine addition to the 3′ end of RNA (RNA uridylation) is a post-transcriptional modification associated with a variety of coding and noncoding RNA substrates across eukaryotes. Fast growing evidence shows that RNA uridylation contributes to the regulation of gene expression in many different specific ways, such as triggering RNA degradation, facilitating RNA processing, or maintaining RNA stability [1,2]. Two recent studies reveal new intriguing roles of uridylation in immune defense against viruses and retrotransposons [[3], [4], [5]]. More impacts of RNA uridylation on biological processes are waiting to be revealed.

RNA uridylation is catalyzed by terminal uridylyl transferases (TUTases), which belong to the DNA polymerase β superfamily. All TUTases contain two canonical domains, a Polβ nucleotidyltransferase domain at the N-terminal side and a poly(A) polymerase-associated domain (PAP) at the C-terminal side, to constitute the core catalytic region [[6], [7], [8]]. In Arabidopsis, two TUTase, HEN1 SUPPRESSOR1 (HESO1) and UTP: RNA uridylyltransferase (URT1), have been experimentally characterized [9]. For microRNAs (miRNAs), HESO1 has been proven to be the major enzyme responsible for RNA uridylation while URT1 contributes to 3′ uridine addition of miRNAs as the TUTase with the second highest impact, and both enzymes function sequentially and cooperatively to add uridine tail for RNA substrates [10,11]. For mRNAs, however, URT1 become the main TUTase to cover the majority of mRNA uridylation (70–80%) and is capable of restoring the size distribution of deadenylated mRNA tails centered at 16 nt HESO1, on the other hand, targets mostly the mRNAs with very short oligo(A) tails and fails to extend them to a sufficient length that allows PABP binding [9,12]. The above evidence reveals an intriguing phenomenon in Arabidopsis: two TUTases, HESO1 and URT1, always collaborate in catalyzing the uridylation of RNA substrates but their significances switch seemingly depending on the style of the RNA substrates. Considering the genomes of the vast majority of plant species encoding both URT1 and HESO1 homologues [13,14], their cooperatively functional relationship in the metabolism of small RNAs and mRNAs are very likely conserved. In addition, URT1 can only add a few uridines (∼1 to 3 nt) to the 3′ end of miRNAs or siRNAs in Arabidopsis while HESO1 is able to quickly extend the uridine tail to a length of above 30 nt in vitro [10,13,15,16]. Furthermore, it has been reported that URT1 has an evident preference for miRNA substrates ending in a 3′ adenine [10,16], which is apparently in line with its functional role in tailing the mRNAs with short oligo(A) tails and protecting them from excessive deadenylation [12].

The distinctive impacts HESO1 and URT1 have on variable RNA substrates suggest that these two TUTases may possess different recognition and/or catalytic specificity towards the same RNA molecule. The structural determinations of HESO1 and URT1 will definitely help illustrate the underlying mechanisms for RNA recognition and uridylation of these two TUTases as well as their functions. In this research, we determined the crystal structures of URT1 in the absence and presence of UTP. Based on structural and sequence comparisons, our research indicated that URT1 shared a same list of conserved residues for UTP recognition with variable TUTases from yeast to mammalian whereas HESO1 might evolve separately to have a different recognition mechanism for UTP. In addition, our structures also revealed that URT1 N552 might be a crucial residue in recognizing the 3′-end nucleotide of its miRNA substrate. Although more structural researches has to be conducted regarding URT1, HESO1, and their complexes with RNAs, our current findings identified Arabidopsis URT1 as a uridylyl transferase that is structurally and functionally very similar to Schizosaccharomyces pombe Cid1, paving the path towards the illustration of the molecular mechanism of URT1/HESO1 dual TUTases in plants.

Section snippets

Expression and purification

A region of URT1 (residues 410–764) was amplified by PCR from synthetic gene and cloned into pET28 vector. URT1 was overexpressed in E.coli Gold (DE3) cells, grown in LB media at 37 °C until OD600 reached about 1.0, and induced with 0.5 mM β-d-1-thiogalactopyranoside (IPTG) for 24 h at 16 °C. Cells were harvested by centrifugation and resuspended in buffer A (20 mM Bis-Tris, pH 6.0 and 1 M NaCl). Cells were lysed by sonication and centrifuged at 16,000 × g for 30 min at 4 °C. The supernatant

URT1 catalytic domain alone is functional in tailing miR158a

URT1, previously identified as a functional paralog of HESO1, was reported to work collaboratively with HESO1 in Arabidopsis in uridylating some miRNAs [10,16]. It has been proven that URT1 and HESO1 act sequentially on some miRNAs, with URT1 mono-uridylating the miRNAs followed by their further uridylation by HESO1. To confirm the nucleotidyl transferase activity of URT1 towards miRNAs, we expressed and purified the core catalytic domain of URT1 (residues 410–764), which was further incubated

Declaration of competing interest

None declared.

Acknowledgements

This work was financially supported by the grants from the Ministry of science and technology of China (2019YFA0508403, 2016YFA0500700), and the Strategic Priority Research Program of the Chinese Academy of Sciences (XDB39000000), the National Natural Science Foundation of China (grant 91940302, 31770805, and 31870760). We thank the staff of Beamline BL19U1 at SSRF for assistance in data collection.

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