The new LAW of microRNA-mediated gene silencing

MicroRNAs (miRNAs) are a class of small RNAs within all the cells of our body that have recently been found to inhibit gene expression. Their mechanism of action is far reaching and complex; each miRNA may control many genes and it is estimated that miRNAs regulate expression of up to 1/3 of all human genes. Human genes are expressed as proteins via a mRNA intermediate which is a copy of the DNA encoded gene in question. This mRNA then travels from the nucleus to the cytoplasm where the code is 'translated' or made into protein and thus expressed. MicroRNAs operate to inhibit gene expression by one of two hypothesised mechanisms: (1) by pairing with a mRNA- and stopping the mRNA being expressed very early on in its translation into protein, initiation block and (2) pairing with target mRNA - stopping the mRNA being expressed in the middle of its translation into protein therefore halting protein production, post-initiation block. In either case the mRNA is not translated into protein but (repressed) but the mRNA remains intact. MicroRNAs show distinct expression patterns in different organisms, cell development stages, and disease models and play an important role in regulating gene expression. Even though we have learnt a great deal about miRNA biology in recent years we still do not know the precise mechanism(s) of how the cell performs miRNA-mediated gene silencing and furthermore how it decides between the different types of miRNA silencing (initiation or post-initiation block). My research group has identified two distinct groups of proteins, (1) called LIMD1, Ajuba, WTIP (LAW) and (2) called Zyxin, LPP, TRIP6 (ZLT). Which we believe may represent the missing components to enable miRNA-directed gene silencing. Furthermore, these two groups of protein may then help the cell decide which type of miRNA silencing to perform. Employ the LAW group and use initiation block, or the ZLT group and induce a post-initiation block. Our research is aimed at determining these possibilities and therefore new biology which will ultimately impact on improved human and animal health in the long term.

MicroRNAs (miRNAs) are small non-coding ~22nt RNAs that play important roles in a wide range of biological processes including development, cellular differentiation, proliferation and apoptosis. To execute their regulatory functions miRNAs assemble together with Argonaute (Ago) family proteins into miRNA induced silencing complexes (miRISCs) which then silenced mRNAs post-transcriptionally. The mechanism by which miRISC regulates translation have been the subject of intense ongoing debate. In recent years there have been significant advances with the identification of key protein components involved in this pathway. However, the complete protein repertoire and mechanism(s) of miRNA-mediated silencing remain to be elucidated. Through BBSRC funding we have revealed for the first time a novel mechanism of regulation of miRNA silencing. We show that a family of three LIM-domain containing proteins LIMD1, Ajuba and WTIP (LAW) are associated with mammalian processing bodies (P-bodies), that LIMD1 is essential for the formation of a specific subset, and that all three family members are critical protein components required for miRNA-mediated gene silencing. Mechanistically, we show that the LAW proteins bind Ago1/2, RCK/p54 and Dcp2 proteins in vivo as well as eIF4E and the mRNA m7GTP cap-protein complex. Specifically, we show that LIMD1 binds both eIF4E and Ago2 concurrently to enhance their association in vivo. We therefore propose that LAW enable miRNA-mediated silencing by facilitating/stabilising a simultaneous association between the translationally repressed m7GTP cap structure and an active miRISC complex attached to the 3'-UTR of mRNA.

The beneficiaries from this research can be clearly divided into three groups. Firstly those in the academic arena where microRNA-mediated gene silencing has become an important cellular pathway for both clinical and non-clinical research. Furthermore, when one considered the degree and variety of cellular processes and diseases pathologies shown to be regulated by microRNA silencing we can begin to understand the profound impact such work will have. Continuing on with disease pathogenesis leads into to the second group of beneficiaries who it is hoped will benefit from this research in the long term. That is, those patients suffering from diseases that are now clearly inked to dysfunction of the microRNA-regulatory pathway such as, those suffering heat disease, cancer and neurodegenerative disorders. The more we understand about the mechanism of microRNA function, in this case LIMD1 related biology, the better we can design novel treatments and therapies to combat these pathologies. Thirdly, the commercial sector will also benefit. We have shown that by regulating microRNA silencing LIMD1 is able to control stem cell differentiation. This specific discovery has resulted in the filing of a Patent for the use of LIMD1 depletion as a novel and highly efficient new methodology/tool for creating induced pluripotent stem cells. This could help realise the potential for reprogramming autologous somatic cells for patient therapy in the near future. Patent PCT/GB09/19773.2. We wish to licence this Patent to the Health Care Industry the benefits of which will be in the fields of regenerative medicine and tissue engineering. Again here the follow on benefits to long term patient health are also clear. With respect to engaging this research and communicating it so as to ensure that benefit is reached can almost be guaranteed once large Pharmacy or health care industries are involved. With the endpoint being to make available related therapies or treatments. Such industries have tremendous resources which can be quickly mobilised to maximise, advertisement, public communication/engagement and exploitation.