Role and function of the ribonuclease Dicer
Relationship between microRNAs and human diseases

In mammals, genes are transcribed into messenger RNAs (mRNAs), which are then translated into proteins. This “simplified” central dogma in molecular biology has become increasingly complex over the past decades. The most recent gain in complexity came from the discovery of a new class of regulatory RNAs, as small as ~21 to 24 nucleotides (nt) in length, known as microRNAs (miRNAs). These RNA species are generated upon successive processing of stem-loop structured primary miRNAs (pri-miRNAs) and miRNA precursors (pre-miRNAs) by the ribonucleases (RNases) III Drosha and Dicer, respectively.

The biological role of miRNAs is linked mainly to their ability to repress mRNA translation through recognition of specific miRNA binding sites usually located in the 3’ nontranslated region. Likely ranging in the thousands in number, a recent study reported that miRNAs may regulate up to 92% of the genes in human! These observations suggest that every cellular processes may be under miRNA control in the human body. Loss of specific miRNA control of gene expression is thus expected to underlie several genetic disorders, most of which remain to be identified.

From Ouellet et al., J. Biomed. Biotechnol. 2006 (2006) 69616

While investigating posttranscriptional gene silencing (PTGS) as a natural antiviral defense mechanism, Hamilton and Baulcombe observed the presence of antisense viral RNA of ~25 nucleotides (nt) in virus-infected plants. These small RNAs were later found to originate from viral double-stranded RNA (dsRNA) processing by Dicer. Since then, several studies have reported a role for RNA silencing in host defense mechanisms against viruses in plants , and recent reports suggest that it may play a similar role in human.

The purpose of this project is to determine the role and importance of Dicer in host defense mechanisms against viruses, such as human immunodeficiency virus type 1 (HIV-1), by elucidating the nature of their interaction, which is likely to be complex and multifaceted. Expected to offer a new perspective on HIV-1 pathogenesis and therapeutics, our findings may shed new light on the ongoing battle between HIV-1 and Dicer that may be constantly shaping the antiviral functionalities of the host RNA silencing machinery.

This project is supported by the Canadian Institutes
of Health Research (CIHR).

From Provost et al., Virus Res. 121 (2006) 107–115

Alzheimer’s disease (AD) currently affects ~2% of the population in industrialized countries, and its incidence is predicted to increase 3-fold within the next 50 years. AD is a slowly progressing neurological disease affecting cholinergic neurons which is caused by the formation of ß-amyloid plaques in specific regions of the brain. An enzyme, called ß-site amyloid precursor protein-cleaving enzyme (Bace), contributes to the formation of these plaques. Intriguingly, post-mortem analyses showed higher ß-amyloid precursor protein converting enzyme (BACE) protein, but not mRNA, levels in the brain cortexes of patients that suffered from Alzheimer's disease (AD), indicative of a loss of Bace mRNA regulation.

In this project, we are investigating the role and importance of two potential Bace miRNAs in order to determine whether the disease could be linked to a dysfunctional gene regulation mediated by miRNAs. Our results may provide the molecular basis underlying BACE1 deregulation in AD and offer new perspectives on the etiology of this neurological disorder.

This project is supported by NARSAD:
The Mental Health Research Association.

MiRNA-guided messenger RNA (mRNA) translational repression is believed to be mediated by effector miRNA-containing ribonucleoprotein (miRNP) complexes harboring FMRP. Clinically, loss of the FMR1 gene product FMRP is the etiologic factor of the fragile X syndrome, the most frequent cause of inherited mental retardation. Together, these observations suggest a possible causal link between the loss of FMRP function in miRNA-guided RNA silencing and the fragile X syndrome.

How miRNAs and single-stranded (ss) small interfering RNAs (siRNAs) are used by the effector RNP complexes for recognition and targeting of specific mRNAs remains obscure. However, rather than being the result of a passive hybridization reaction, formation of a miRNA:mRNA or ss siRNA:mRNA transition complex is more likely to be facilitated by a component of the miRNP or siRNP complexes.

Recently, we have shown that FMRP can act as a miRNA acceptor protein for Dicer and facilitate assembly of miRNAs on specific target RNA sequences. Functioning within a duplex miRNP, FMRP may also mediate mRNA targeting through a strand exchange mechanism, in which the miRNA* of the duplex is swapped for the mRNA. The hypothesis that FMRP can exert a dual role in mediating and relieving miRNA-guided translational repression is attractive. Suboptimal utilization of miRNAs may thus account for some of the molecular defects in patients with the fragile X syndrome.

This project is supported by the Canadian Institutes of Health Research (CIHR).

From Plante and Provost, J. Biomed. Biotechnol. 2006 (2006) 16806

Dicer is the RNase III responsible for the conversion of pre-miRNAs into mature ~21-24 nt miRNAs. This multi-domain enzyme has been proposed to function by the intramolecular dimerization of its two RNase III domains, like Drosha, assisted by the PIWI/Argonaute/Zwille (PAZ) and the C-terminal dsRNA binding domain (dsRBD). Its central PAZ domain specifically recognizes the 2-nt 3’ overhang present in the pre-miRNA and generated upon cleavage by Drosha. The PAZ and RNase IIIa domains may then act as a caliper in generating similarly-sized miRNAs. Despite its central importance in miRNA biogenesis, the cellular context in which Dicer functions in RNA silencing remains poorly understood.

In this research program, we wish to improve our understanding of the biological role, function and regulation of human Dicer through the elucidation of the complete Dicer protein interaction network. Our results may provide key insights into genetic diseases possibly linked to alterations in Dicer expression or function.

This project is supported by the Natural Sciences
and Engineering Research Council of Canada (NSERC).