Structure and mechanism of CutA, RNA nucleotidyl transferase with an unusual preference for cytosine.

Deepshikha Malik,Paweł Krawczyk,Rafał Tomecki,Agnieszka Napiórkowska,Kamil Kobyłecki,Andrzej Dziembowski,Aleksandra Jakielaszek,Marcin Nowotny,Jarosław Poznański

doi:10.1093/nar/gkaa647

Abstract

Template-independent terminal ribonucleotide transferases (TENTs) catalyze the addition of nucleotide monophosphates to the 3′-end of RNA molecules regulating their fate. TENTs include poly(U) polymerases (PUPs) with a subgroup of 3′ CUCU-tagging enzymes, such as CutA in Aspergillus nidulans. CutA preferentially incorporates cytosines, processively polymerizes only adenosines and does not incorporate or extend guanosines. The basis of this peculiar specificity remains to be established. Here, we describe crystal structures of the catalytic core of CutA in complex with an incoming non-hydrolyzable CTP analog and an RNA with three adenosines, along with biochemical characterization of the enzyme. The binding of GTP or a primer with terminal guanosine is predicted to induce clashes between 2-NH2 of the guanine and protein, which would explain why CutA is unable to use these ligands as substrates. Processive adenosine polymerization likely results from the preferential binding of a primer ending with at least two adenosines. Intriguingly, we found that the affinities of CutA for the CTP and UTP are very similar and the structures did not reveal any apparent elements for specific NTP binding. Thus, the properties of CutA likely result from an interplay between several factors, which may include a conformational dynamic process of NTP recognition.

Highlights

Template-independent terminal ribonucleotide transferases (TENTs) [1] catalyze the addition of nucleotide monophosphates to the 3 -end of various RNA molecules and such additions play an important role in determining fate of these RNAs [2]
The primary aim of the present study was to solve crystal structures of CutA to understand the molecular basis of its unique biochemical properties, including (i) processive RNA polymerization exclusively for adenosines, (ii) a complete lack of activity for guanosine triphosphate (GTP) as the incoming nucleotide, (iii) an inability to extend primers that end with guanosines and (iv) a preference for incoming cytidines over uridines in the nucleotide transfer reaction
We sought to elucidate the structural basis of several key unique features of CutA, including processive RNA polymerization exclusively for adenines, a complete lack of activity for GTP as the incoming nucleotide, an inability to extend primers that end with guanosines, and a preference for the incorporation of cytidines over uridines

Summary

Introduction

Template-independent terminal ribonucleotide transferases (TENTs) [1] catalyze the addition of nucleotide monophosphates to the 3 -end of various RNA molecules and such additions play an important role in determining fate of these RNAs [2]. A growing number of so-called poly(U) polymerases (PUPs), termed terminal uridyl transferases (TUTases), which mostly add uridyl ribonucleotides to the 3 end of RNA, have recently been described These are involved in multiple pathways of RNA regulation but usually induce the accelerated decay of target RNA molecules [6,7] through a mechanism in which uridine tagging at the 3 -end of a transcript by a TUTase is followed by decapping and 5 -3 or 3 -5 exonucleolytic degradation [8,9,10]. Addition of a short, up to 7 nt long, heteropolymeric C/Urich tail (3 CUCU-tagging) has been observed in filamentous fungi, such as Aspergillus nidulans [14,15] Addition of such C/U extensions to the poly(A) tail of mRNA leads to rapid decay [14]. Tails with atypical composition are likely added to mRNAs in other organisms [16,17], but little is known about the mechanistic and structural basis of these unusual enzymatic activities

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Conclusion