T4 RNA Ligase 2 truncated active site mutants: improved tools for RNA analysis

Sebastien Viollet,Gregory B Robb,Fanglei Zhuang,Ryan T Fuchs,Daniela B Munafo

doi:10.1186/1472-6750-11-72

Abstract

BackgroundT4 RNA ligases 1 and 2 are useful tools for RNA analysis. Their use upstream of RNA analyses such as high-throughput RNA sequencing and microarrays has recently increased their importance. The truncated form of T4 RNA ligase 2, comprising amino acids 1-249 (T4 Rnl2tr), is an attractive tool for attachment of adapters or labels to RNA 3'-ends. Compared to T4 RNA ligase 1, T4 Rnl2tr has a decreased ability to ligate 5'-PO4 ends in single-stranded RNA ligations, and compared to the full-length T4 Rnl2, the T4 Rnl2tr has an increased activity for joining 5'-adenylated adapters to RNA 3'-ends. The combination of these properties allows adapter attachment to RNA 3'-ends with reduced circularization and concatemerization of substrate RNA.ResultsWith the aim of further reducing unwanted side ligation products, we substituted active site residues, known to be important for adenylyltransferase steps of the ligation reaction, in the context of T4 Rnl2tr. We characterized the variant ligases for the formation of unwanted ligation side products and for activity in the strand-joining reaction.ConclusionsOur data demonstrate that lysine 227 is a key residue facilitating adenylyl transfer from adenylated ligation donor substrates to the ligase. This reversal of the second step of the ligation reaction correlates with the formation of unwanted ligation products. Thus, T4 Rn2tr mutants containing the K227Q mutation are useful for reducing undesired ligation products. We furthermore report optimal conditions for the use of these improved T4 Rnl2tr variants.

Highlights

T4 RNA ligases 1 and 2 are useful tools for RNA analysis
In addition to products of the expected size, we detected ligation products that migrated with longer apparent length when treated with Rnl1, Rnl2, or Rnl2tr (Figure 2). This finding was surprising since our ligation reaction conditions should have prevented concatamerization of RNA substrates
In this work, we introduced conservative active-site mutations in T4 Rnl2tr to determine their characteristics, especially as they relate to formation of unwanted ligation products

Summary

Introduction

T4 RNA ligases 1 and 2 are useful tools for RNA analysis. Compared to T4 RNA ligase 1, T4 Rnl2tr has a decreased ability to ligate 5’-PO4 ends in singlestranded RNA ligations, and compared to the full-length T4 Rnl, the T4 Rnl2tr has an increased activity for joining 5’-adenylated adapters to RNA 3’-ends. The combination of these properties allows adapter attachment to RNA 3’ends with reduced circularization and concatemerization of substrate RNA. The T4 RNA ligases are able to join adjacent 3’-OH and 5’-PO4 polynucleotides as a result of a three-step reaction (Figure 1). The reaction is promoted by the attack on the 5’-phosphorus by the 3’-OH and results in AMP release [4]

Methods

Results

Discussion

Conclusion