Introducing RNA Ligase

What is the Function of theRNA Ligase ?

Eukaryotic RNA precursors undergo a process of splicing and ligation to form mature functional RNA molecules. RNA ligases are involved in repair, splicing, and editing pathways, which either reseal broken RNA or alter its primary structure. RNA ligases play a crucial role in RNA sealing processes, primarily involved in tRNA repair, mRNA splicing, and the biological generation of circular RNA. The catalytic steps of 5'-3' RNA ligases mainly involve three steps: the enzyme's ATP-dependent self-adenylation, transfer of AMP to the 5'-PO4 to form an AppRNA intermediate, and the final step of attack by the 3'-terminal hydroxyl group on the phosphate, thereby making 5'-3' RNA ligases widely applicable in RNA editing and sequencing.

View of interphase nuclei with the mRNA, DNA and Ligase.

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RNA Ligase vs DNA Ligase

DNA ligase and RNA ligase are enzymes that play important roles in cells. They both catalyze the joining of broken DNA or RNA strands. DNA ligase primarily acts on DNA molecules, while RNA ligase mainly acts on RNA molecules. DNA ligase plays a crucial role in DNA replication and repair processes, ensuring the integrity and stability of DNA. RNA ligase, on the other hand, functions in RNA repair and splicing, helping to maintain the structure and function of RNA. The main difference between RNA ligase and DNA ligase is that RNA ligase uses ssRNA molecules to arrange and connect, while DNA ligase requires double-stranded structures.

What are the Different Types of RNA Ligase?

T4 RNA Ligase 1 (ssRNA Ligase)

T4 RNA Ligase 1 (ssRNA Ligase) is an ATP-dependent enzyme that catalyzes the formation of phosphodiester bonds between the 5'-P end and the 3'-OH end of single-stranded RNA, single-stranded DNA, or single nucleotide molecules, either intermolecularly or intramolecularly. T4 RNA Ligase 1 exhibits the highest efficiency in joining RNA molecules, followed by joining DNA to RNA, and the lowest efficiency in joining DNA molecules together, thus also being considered an ssRNA ligase.

T4 RNA Ligase 2 (dsRNA Ligase)

T4 RNA Ligase 2 (dsRNA Ligase) is an ATP-dependent enzyme specialized in joining double-stranded RNA (dsRNA) ends. It can also be used to link the 3' hydroxyl group of RNA within a double-stranded structure to the 5' phosphate of DNA. This enzyme requires the presence of a 5' phosphate and a 3' hydroxyl group for ligation, enabling the connection reaction between the 5' phosphate of RNA or DNA chains and the 3' hydroxyl group of RNA chains. T4 RNA Ligase 2 can be utilized for intramolecular circularization and intermolecular linearization of double-stranded RNA molecules. During ligation, the enzyme relies on the presence of a 5' phosphate and a 3' hydroxyl group, facilitating the connection reaction between the 5' phosphate of RNA or DNA chains and the 3' hydroxyl group of RNA chains. A distinctive feature of T4 RNA Ligase 2 compared to T4 RNA Ligase 1 is its significantly higher activity in joining nicks in dsRNA than in linking ssRNA ends. Hence, for intramolecular circularization of RNA molecules facilitated by RNA secondary structures, T4 RNA Ligase 2 is commonly preferred.

T4 RNA Ligase 2, Truncated KQ

T4 RNA Ligase 2, Truncated KQ, specifically joins the pre-adenylated 5' end of DNA or RNA to the 3' end of RNA. This enzyme does not require ATP for ligation but necessitates pre-adenylated substrates. Unlike the full-length ligase, this enzyme is unable to link the phosphorylated 5' end of RNA or DNA to the 3' end of RNA. Also known as Rnl2, it has been employed to optimize adapter ligation in microRNA cloning, reducing background ligation by utilizing pre-adenylated adapters exclusively. This ligase can attach single-stranded adenylylated DNA or RNA (App-DNA or App-RNA) oligonucleotides to small RNAs. The pre-adenylated 5' end of DNA or RNA is linked to the 3' end of RNA without the need for ATP, preventing cyclization and other undesirable bimolecular reactions. It is used for preparing cDNA libraries for small RNA transcriptome analysis (e.g., RNA-Seq) and optimal adapter ligation for miRNA cloning.

RtcB Ligase

The RtcB ligase from Escherichia coli joins single-stranded RNA molecules possessing a 3'-phosphate or a 2',3'-cyclic phosphate to another RNA molecule with a 5'-hydroxyl group. This ligation process requires GTP and MnCl₂ and proceeds via a 3'-nucleotide intermediate.

CircLigase II ssDNA Ligase

This thermostable ligase catalyzes intramolecular ligation (i.e., circularization) of ssDNA templates >15 nucleotides long with a 5' phosphate and a 3' hydroxyl group. It ligates the ends of ssDNA without complementary sequences. Under standard reaction conditions, no detectable single-stranded DNA multimers or multimeric DNA circles are produced. Due to its high adenylylation efficiency, a single CircLigase II enzyme can only ligate a single non-adenylylated DNA molecule, and the reaction stops in the absence of ATP. Therefore, a 1:1 stoichiometry of ligase to substrate is required to drive the ligation reaction to completion. This ligase is utilized with ssDNA templates for rolling-circle replication or rolling-circle transcription experiments and for RNA polymerase and RNA polymerase inhibitor assays.

CircLigase ssDNA Ligase

This thermostable and ATP-dependent ligase catalyzes intramolecular ligation (i.e., circularization) of ssDNA templates >30 nucleotides long with a 5' phosphate and a 3' hydroxyl group. It ligates the ends of ssDNA without complementary sequences. Under standard reaction conditions, no detectable single-stranded DNA multimers or multimeric DNA circles are produced. Due to its low adenylylation efficiency, CircLigase enzyme exhibits a high turnover rate. It can reversibly and repetitively act on multiple pre-adenylated DNA molecules under non-stoichiometric reaction conditions. This ligase is primarily used with ssDNA templates for rolling-circle replication or rolling-circle transcription experiments and for RNA polymerase and RNA polymerase inhibitor assays.

ssRNA Cyclase and ssDNA/RNA Cyclization Ligase

Both of these cyclization ligases are thermostable enzymes capable of catalyzing the intramolecular ligation of single-stranded DNA (ssDNA) and single-stranded RNA (ssRNA) substrates, resulting in the formation of cyclic molecules. In the cyclization reaction, it is required that the catalytic substrates of ssDNA/RNA possess a 5'-phosphate group and a 3'-OH group.