RNA Editing

RNA editing or post-transcriptional editing are mechanisms that the mRNA molecules undergo to be able to function correctly in the cell. There are two unique types of post-transcriptional RNA editing that occurs in the genus Trypanosoma. These are uridine insertion or deletion in the mitochondria, and the trans splicing of the spliced leader RNA onto chromosomal mRNA.

Uridine Insertion or Deletion

RNA molecules are made up of four different nucleotides; adenine, guanine, cytosine and uridine. In trypanosomes, some of the DNA that is transcribed is incomplete. The mRNA that is formed sometimes has too many or too few uridine nucleotides in it. In order for these mRNA molecules to be able to function correctly, the Trypanosome needs a way to add or delete the extra uridine nucleotides. This process occurs only in the mitochondria. The incomplete DNA is called a cryptogram.

In the mitochondria, the DNA is folded into structures called minicircles and maxicircles. The minicircles encode for a specific type of RNA called guide RNAs or gRNAs. These guide RNAs are used in uridine insertion and deletion. They bind to the DNA and tell where uridine nucleotides need to be inserted or deleted. Guide RNAs are complementary to short stretches of the pre-mRNA molecule. There is a short number of nucleotides at the start of the guide RNA that anchors the guide RNAto the mRNA. The maxicircles contain the genes that need to be edited.

Guide RNA’s bind to the mRNA, creating a hybrid. Then a gRNA-dependent endonuclease cuts the mRNA at the first place where the mRNA and the gRNA don’t match. If a uridine needs to be added, then a 3’-terminal uridylyl transferase or a TUTase adds however many uridines to the 3’ end that are needed. An RNA ligase comes along and ligates the two fragments together. If instead of a uridine needing to be added, it needs to be deleted, then an exonuclease cuts out the extra uridine. Then an RNA ligase comes in and reattaches the two fragments. (See figure 1)

It has been found that there is a multi-protein complex that is thought to contain at least the RNA ligases. It is known that two proteins, TbMp52, and TbMp48 are the two RNA ligases within this multi-protein complex. It is thought that this multi-protein complex binds to the gRNA-mRNA hybrid. It is also thought that the other proteins used in this machine bind to this multiprotein complex, but that is not known for sure.

This type of RNA editing is important for the continued survival of the bloodform of the organism, because NADH dehydrogenase subunits are produced and edited in the mitochondria, and are needed for making ATP in the bloodform of Trypanosoma brucei. It is thought that this type of RNA editing may provide chemotherapeutic targets for Trypanosomes.

Trans splicing

It has been found that all mRNA molecules have an additional 35 to 39 nucleotides added to the 5’ end of the transcribed RNA molecule. These extra nucleotides are added during a unique mechanism of RNA editing called trans splicing. The extra 35 nucleotides are called the spliced leader RNA or SL RNA. Trans splicing is a special type of capping unique to trypanosomes. The cap that is attached to the mRNA is the spliced leader.

During trans splicing, a small RNA molecule, the spliced leader RNA is added to the mRNA. The mechanism for trans splicing involves an adenosine nucleotide in an intron of the mRNA attaching itself to the intersection between the spliced leader and its intron. The intron of the spliced leader deattaches from the spliced leader, and is attached to the intron of the mRNA. The 3’ end of the spliced leader then attacks the intersection between the mRNA and it intron. The two introns are displaced by the spliced leader. This process occurs within the nucleus.

The spliced leader RNA is encoded for by a specific gene in the genome. The spliced leader or SL is derived from the 5’ end of a small nuclear RNA or snRNA. The small nuclear RNA is about 120 nucleotides long. Sometimes it can be as long as 140 nucleotides, or as short as 60 nucleotides long. The mRNA of this gene is composed of the spliced leader and what appears to be an intron. The intron is displaced by the mRNA during trans splicing.

In order for the spliced leader to be trans spliced onto the mRNA, a cap has to be formed at the 5' end of the spliced leader. This cap is formed through a process by which nucleotides are added and methylated. First a 7-methyl guanine is added to the 5’ end of the SL RNA by a 5’-5’ phosphate bond. This is followed by the addition of four methylated nucleotides. This forms the cap: m7G(5’)ppp(5’)- N6,N6,2’-O-trimethyladenosine-p-2’O-methyladenosine-p-2’-O-methylcytosine-p-N3,2’-O-dimethyluridine. This structure is called a cap 4 structure. It is thought that the capping of the SL RNA occurs while the SL RNA is being transcribed. The mechanism for forming the cap is processive, meaning that specific substrates have to be added before others can be attached.

The cap of the spliced leader is essential in directing mRNAs to the various pathways that process and transport mRNA molecules outside of the cell nucleus. It also regulates mRNA translation initiation. The cap is also essential in allowing the SL RNA to be used in trans splicing.

The amount of modification that occurs depends on the length of the spliced leader RNA. If the spliced leader RNA is between 56 and 111 nucleotides in length then the modification that occurs is only partial. This means that only two modified adenosine nucleotides are added. If the spliced leader is longer than that then either partial or full modification occurs.

The addition of the cap may also depend upon a spliced leader RNP. A spliced leader RNP is a spliced leader RNA molecule assembled in to a ribonuclear protein or RNP. Whether or not the spliced leader RNA bonds to the SL RNP depends upon the length of the spliced leader RNA molecule.