The spliceosome is
crucially important for the proper processing of pre-mRNA into fully
functioning mature mRNA. The spliceosome is a structure composed of proteins and
a number of snRNPs. However, the concentrations of each snRNP are not the same,
with a drastically higher level of the U1 snRNP present within cells. This
paper therefore investigated the potential role of this snRNP, and found that
its role in splicing is not its sole function, with evidence suggesting that it
is also involved in the protection of pre-mRNA, thus preventing its premature
processing. This research could have implications on the human disease spinal
muscular atrophy, which is known to be caused by changing concentrations of
snRNPs.
Ribonucleic acid (RNA) is a class
of biological molecules which are crucially important for a vast number of
functions, including the proper regulation and expression of genes. RNA is
further divided into further categories, one of which includes messenger RNA
(mRNA), which is used in cellular organisms to convey genetic information and
allow the synthesis of the specific proteins required for an organism’s
survival. However, prior to the production of mature mRNA, primary transcripts
(pre-mRNAs) must undergo post-transcriptional processing such as splicing,
which is carried out by the spliceosome.
The spliceosome is predominantly
assembled from snRNPs as well as a number of other protein complexes, and
functions by removing introns from the pre-mRNA transcript. The U2 spliceosome
is composed of the U1, U2, U4, U6 and U5 snRNPs. It is these snRNPs which
recognize the sequences which define the classes of introns, giving them a
crucial process during splicing reactions.
Whilst snRNPs are crucially
important for the proper assembly of the spliceosome, each snRNP is not present
in equal concentrations. This is highlighted in the case of the U1 snRNP in
which there are thought to be 10 million molecules within each human cell, a
much higher concentration than is observed in other snRNPs.
The full implications on cellular functions that changes in the level of snRNPs could have is not currently known. However, previous research has found that perturbing the biogenesis of snRNPs induces a high level of splicing abnormalities. A greater understanding of the role of snRNP abundance is important, with implications in a number of human diseases, including spinal muscular atrophy which is caused by a reduction in the level of functional SMN protein.
However, there is often
difficultly in researching the varying levels of snRNPs and the effect that
they may have, due to the high disparity that occurs between different cells
depending on their function. Recent research by Kaida et al (2010) therefore wished to further expand knowledge in this
area though investigating the effect of a reduction in the levels of snRNPs
individually. This was tested through the use of antisense
morpholino oligonucleotide (AMO) technology, which uses sequence of
oligonucleotide designed to be complimentary to specific sequences which are
known to be transcribed into mRNA, which stops it from performing the cellular
role it is normally associated with.
AMO was used to mask the 5’ end
of the U1 snRNA, which is the region associated with the identification of
splicing sites, thus preventing its normal function. The action of AMO was seen
to be dose dependent, with a concentration of 7.5µM U1 AMO finally used, as
this level deemed the 5’ site completely inaccessible. Treatment with the
general splicing inhibitor SSA was also used as a reference to understand the
U1 snRNPs full effect.
The results from this treatment
suggested a role for U1 snRNP unrelated to its normal role in splicing. It was
found that, when the U1 snRNP function was knocked out a much higher
concentration of prematurely cleaved and polyadenylated pre-mRNAs could be
observed. This suggests that this snRNP is capable of protecting pre-mRNA transcripts
from premature cleavage and polyadenylation (PCPA). Whilst the mechanism
through which this type of protection is provided is not currently known,
several models have been proposed (explained in fig 1). One method suggests it
is believed that U1 prevents this activity through masking sites with potential
polyadenylation signals (PAS), making them unavailable for their function of
pre-mRNA processing. This dual action could also go some way to explaining why
such high levels can be observed within cells, owing to the fact that they may
be needed in separate locations along a genome simultaneously.
However, what can be conferred
with a high level of certainty is that U1 must be used to modulate the level of
gene expression within a cell, by defining the numbers of pre-mRNA transcripts
which can reach maturity and affect the protein production of the cell.
It may therefore be an important
step in the future research of this work to fully understand the mechanism
through which U1 confers protection, therefore potentially allowing us to
artificially control its function both in normal patients, as well as those
suffering from diseases where gene expression induces a negative phenotype.
