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Post-transcriptional modification,
otherwise known as RNA editing, is a mechanism that the transcribed
mRNA molecules undergo to be able to leave the nucleus and
function correctly in the cell. In the organism Trypanosoma
brucei, there are two unique types of post-transcriptional
modification that occurs, along with the regular post-transcriptional
modifications. These modifications 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 either 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 either add or delete the extra
uridine nucleotides. This incomplete DNA is called a cryptogram.
This process occurs only in the mitochondria.
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 while the maxicircles encode for the genes that
need to be edited. These guide RNA molecules are used in uridine
insertion and deletion. They bind to the pre-mRNA and tell
where uridine nucleotides need to be inserted or deleted.
(Weaver 2002) Guide RNAs are complementary to short stretches
of the pre-mRNA molecule. They are about 60 kilobases in length.(Kable
et al. 1996) There
is a short number of nucleotides at the start of the guide
RNA that anchors the guide RNAto the mRNA. The guide RNA also
has a 3' u-tail. It links to an 11-nucleotide purine rich
sequence that is to the 5' end of the edited region, hypothetically
to help it stabilize the gRNA-mRNA hybrid. (Simpson et al.
2000)
In order for uridine insertion
or deletion to occur, several different proteins are needed
in addition to the guide RNA. These proteins include a protein
that cuts the mRNA into fragments, a gRNA-dependent riboendonuclease,
a protein that inserts a uridine nucleotide, a terminal uridylyl
transferase, a different protein that takes out a uridine
nucleotide, an exonuclease, and a protein that connects the
two fragments together again an RNA ligase. (Simpson et al.
2003)
There are two RNA ligases
found within the mitochondria, these are both found within
a multi-protein complex that can only interact with the gRNA-mRNA
hybrid. The two RNA ligases are known as TbMp52 and TbMp48.
It is thought that these two proteins either have different
functions in the mitochondria or they function during different
times in the lifecycle. (Schnaufer et al. 2001) It is also
thought that the other proteins used in this machine bind
to this multiprotein complex, but that is not known for sure.
(Simpson, Sbicego and Aphasizhev 2003)
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 the
adenine nucleotide in the guideRNA doesn't match the nucleotide
in the mRNA, but if a uridine needs to be deleted, then the
uridine nucleotide in the mRNA molecule doesn't match the
nucleotide in the guideRNA. 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. Uridine
insertion/deletion occurs in the 3' to 5' direction. (Simpson
et al. 2003) (See figure 1)
This type of RNA editing
is important for the survival of the bloodstream form of Trypanosoma
brucei, because many proteins are transcribed in the
mitochondria that are needed for the survival of the trypanosome.
These proteins include rotenone-sensitive NADH dehydrogenase.
The loss of this protein is lethal to the bloodstream form
of Trypanosoma brucei. It is thought that this type of RNA
editing may provide chemotherapeutic targets for Trypanosomes.
(Schnaufer, et al. 2001)
Trans
splicing
It has been found that all
chromosomal mRNA molecules have an additional 35 to 39 nucleotides
added to the 5’ end. 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.
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, molecule. 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. (Mair, Ullu, & Tschudi,
2000) The mRNA of this gene is composed of the spliced leader
and what appears to be an intron. The spliced leader also
contains a specific cap structure of its own. The intron is
displaced by the mRNA during trans splicing. It appears that
all mRNA molecules start with an intron which is displaced
by the spliced leader. (Weaver, 2002)
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 attaches itself 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 combined
introns are displaced by the spliced leader. (see
figure 2) This process occurs within the nucleus. (Weaver,
2002)
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 methylated and then attached to the 5' end of the spliced
leader. (Ullu &Tschudi, 1991) 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 more 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. (Mair et al., 2000)
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 into 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. (Mair et al., 2000)
The addition of the spliced
leader RNA is important because 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. (Mair et al., 2000)
Works cited
Kable, M., Seiwert, S., Heidmann, S. and Stuart, K. (1996).
RNA editing: A mechanism for gRNA-specified uridylate insertion
into precursor mRNA. Science 273: 1189-1195.
Mair, G., Ulllu, E., and Tschudi, C. (2000). Cotranscriptional
cap 4 formation on the Trypanosoma brucei spliced
leader RNA. Journal of Biological Chemistry 275:
28994-28999.
Schnaufer, A., Panigrahi, A.K., Panicucci, B., Igo, R.P.,
Salavati, R., Stuart, K. (2001). An RNA ligase essential for
RNA editing and survival of the bloodstream form of Trypanosoma
brucei. Science 291: 2159-2162.
Simpson, L., Sbicego, S., and Aphasizhev, R. (2003). Uridine
insertion/deletion RNA editing in trypanosome mitochondria:
A complex business. RNA 9: 265-276.
Simpson, L., Thiemann, O., Savill, N., Alfonzo, J., and Maslov,
D. (2000). Evolution of RNA editing in trypanosome mitochondria.
Proceedings of the National Academy of Sciences USA
97: 6986-6993.
Ullu, E., and Tschudi, C. (1991). Trans splicing in trypanosomes
requires methylation of the 5' end of the spliced leader RNA.
Proceedings of the National Academy of Sciences USA
88: 10074-10078.
Weaver, R. (2002) Molecular Biology. New York, NY:
McGraw Hill
Created by Erin Sargent as part of a biology
senior seminar at Earlham College
Last updated: April 23, 2003 |
