Final Report Summary - MIRT (Elucidation of a microRNA turnover machinery in C. elegans)
Summary and overview of the results
MicroRNAs (miRNAs) constitute a large class of genes that control diverse biological processes. Animal miRNAs repress their targets through an antisense mechanism, where they base-pair imperfectly with their target mRNAs, promoting translational repression and target degradation. Recent studies have linked over- and underexpression of miRNAs to various human diseases, particularly cancers, where miRNAs can function as tumour suppressors and oncogenes. Accordingly, miRNA expression is a highly regulated process under physiological conditions that is controlled at the level of transcription and, post-transcriptionally, at various steps of miRNA maturation. Such tight regulation helps to maintain constant steady-state levels of some miRNAs during animal development, and dynamic expression patterns of others.
As RNA concentrations are generally a function of biogenesis and turnover, it is possible that active miRNA degradation can also modulate miRNA accumulation, providing an additional layer of regulation of miRNA activity. Thus understanding of miRNA turnover would not only provide new insights into the miRNA metabolism circuit but might also open up new avenues towards unravelling of pathological states associated with miRNA dysregulation. My first objective was to identify a miRNA turnover machinery/machinery component(s) employing genetics and the second being biochemical characterisation of miRNA turnover and its machinery/machinery component(s).
Based on my results obtained during the last 24 months (1st March 2008-28th February 2010) I report here that alongside regulation at the level of biogenesis, functional miRNA homeostasis is further regulated by degradation of mature miRNAs in vivo, mediated by the XRN-2; an exoribonuclease of C. elegans, which was identified through a genetic screen. The XRN-2-dependent miRNA turnover can be recapitulated in larval lysates, where processing of pre-let-7 by Dicer, unannealing of the let-7 duplex, and loading into Argonaute proteins are coupled processes that precede degradation of the mature miRNA. Unexpectedly, although Argonaute:miRNA complexes are highly salt-resistant, larval lysate promotes efficient release of the miRNA, exposing it to degradation by XRN-2. Release and degradation can both be blocked by addition of miRNA target RNA. My results thus suggest the presence of an additional layer of regulation of miRNA activity that might be important for rapid changes of miRNA expression profiles during developmental transitions, and maintenance of miRNA steady-state concentrations.
Importantly, although C. elegans has excelled as a model organism through its strength in genetics and cell biology, I use it here to biochemically recapitulate multiple steps of miRNA biogenesis and turnover (pre-miRNA processing by Dicer, unannealing of the resulting miRNA duplex, incorporation of the miRNA guide strand into Argonaute/RISC and release and degradation of the mature miRNA) in a coordinated manner. This demonstration demolishes the view that biochemical approaches are inherently limited when working with C. elegans, opening up profoundly new avenues in C. elegans research in various fields.
Conclusions and socioeconomic impacts of the project
Through my work I have characterised degradation of mature miRNAs by a dedicated nuclease, which is a novel step controlling miRNA homeostasis in C. elegans.
Although miRNAs can be transcriptionally regulated, it is now becoming clear that substantial regulation of miRNA activity occurs downstream of transcription, during biogenesis. However, it has not been known whether degradation of mature miRNAs might affect miRNA levels and activity. Now I show this to be the case and identify a dedicated 'microRNase', XRN-2. I demonstrate that XRN-2 acts on functional miRNA molecules, whose activity it can terminate, excluding a mere 'scavenger' RNase function. Overturning the dogma of stable, static miRNA: Argonaute complexes (a notion entrenched in the literature owing to the resistance of these complexes to high salt concentration), I show that C. elegans cell lysates contain an activity that can efficiently release Arognaute-bound miRNA for degradation by XRN-2. My results thus paint a much more dynamic view of miRNAs than previously anticipated.
Apart from its scientific importance for a better understanding of miRNAs, my work also has implications for the various human diseases that are caused by dysregulation of miRNA expression through mechanisms that remain largely undescribed. Thus, complete understanding of this pathway would significantly affect our perception of miRNA regulatory networks. Most importantly, with aberrant miRNA expression levels now clearly linked to various diseases, particularly cancer, I expect that the advance outcomes of this project will have major medical implications.
MicroRNAs (miRNAs) constitute a large class of genes that control diverse biological processes. Animal miRNAs repress their targets through an antisense mechanism, where they base-pair imperfectly with their target mRNAs, promoting translational repression and target degradation. Recent studies have linked over- and underexpression of miRNAs to various human diseases, particularly cancers, where miRNAs can function as tumour suppressors and oncogenes. Accordingly, miRNA expression is a highly regulated process under physiological conditions that is controlled at the level of transcription and, post-transcriptionally, at various steps of miRNA maturation. Such tight regulation helps to maintain constant steady-state levels of some miRNAs during animal development, and dynamic expression patterns of others.
As RNA concentrations are generally a function of biogenesis and turnover, it is possible that active miRNA degradation can also modulate miRNA accumulation, providing an additional layer of regulation of miRNA activity. Thus understanding of miRNA turnover would not only provide new insights into the miRNA metabolism circuit but might also open up new avenues towards unravelling of pathological states associated with miRNA dysregulation. My first objective was to identify a miRNA turnover machinery/machinery component(s) employing genetics and the second being biochemical characterisation of miRNA turnover and its machinery/machinery component(s).
Based on my results obtained during the last 24 months (1st March 2008-28th February 2010) I report here that alongside regulation at the level of biogenesis, functional miRNA homeostasis is further regulated by degradation of mature miRNAs in vivo, mediated by the XRN-2; an exoribonuclease of C. elegans, which was identified through a genetic screen. The XRN-2-dependent miRNA turnover can be recapitulated in larval lysates, where processing of pre-let-7 by Dicer, unannealing of the let-7 duplex, and loading into Argonaute proteins are coupled processes that precede degradation of the mature miRNA. Unexpectedly, although Argonaute:miRNA complexes are highly salt-resistant, larval lysate promotes efficient release of the miRNA, exposing it to degradation by XRN-2. Release and degradation can both be blocked by addition of miRNA target RNA. My results thus suggest the presence of an additional layer of regulation of miRNA activity that might be important for rapid changes of miRNA expression profiles during developmental transitions, and maintenance of miRNA steady-state concentrations.
Importantly, although C. elegans has excelled as a model organism through its strength in genetics and cell biology, I use it here to biochemically recapitulate multiple steps of miRNA biogenesis and turnover (pre-miRNA processing by Dicer, unannealing of the resulting miRNA duplex, incorporation of the miRNA guide strand into Argonaute/RISC and release and degradation of the mature miRNA) in a coordinated manner. This demonstration demolishes the view that biochemical approaches are inherently limited when working with C. elegans, opening up profoundly new avenues in C. elegans research in various fields.
Conclusions and socioeconomic impacts of the project
Through my work I have characterised degradation of mature miRNAs by a dedicated nuclease, which is a novel step controlling miRNA homeostasis in C. elegans.
Although miRNAs can be transcriptionally regulated, it is now becoming clear that substantial regulation of miRNA activity occurs downstream of transcription, during biogenesis. However, it has not been known whether degradation of mature miRNAs might affect miRNA levels and activity. Now I show this to be the case and identify a dedicated 'microRNase', XRN-2. I demonstrate that XRN-2 acts on functional miRNA molecules, whose activity it can terminate, excluding a mere 'scavenger' RNase function. Overturning the dogma of stable, static miRNA: Argonaute complexes (a notion entrenched in the literature owing to the resistance of these complexes to high salt concentration), I show that C. elegans cell lysates contain an activity that can efficiently release Arognaute-bound miRNA for degradation by XRN-2. My results thus paint a much more dynamic view of miRNAs than previously anticipated.
Apart from its scientific importance for a better understanding of miRNAs, my work also has implications for the various human diseases that are caused by dysregulation of miRNA expression through mechanisms that remain largely undescribed. Thus, complete understanding of this pathway would significantly affect our perception of miRNA regulatory networks. Most importantly, with aberrant miRNA expression levels now clearly linked to various diseases, particularly cancer, I expect that the advance outcomes of this project will have major medical implications.
