smallRNAs Small molecules, big functions

Brief history The first described microRNA, lin-4 was cloned and characterised as a translational repressor of developmental timing from Caenorhabditis. elegans by Lee et al (1993) and Wightman et al (1993). The transcript of this gene was highly unusual as it was non-coding, and produced extremely small transcripts (22nt) from hairpin structured RNA precursors. Second microRNA, let-7 was also cloned from C. elegans (Reinhart et al, 2000). There are currently 474 human cloned and characterised microRNA sequences deposited in the miRBase database ( MicroRNAs primarily function as translational repressors by binding to complementary target sequences in the 3’ UTR (untranslated region) of mRNA.

Brief history Between 10–30% of all human genes are a target for microRNA regulation (John et al, 2004; Lewis et al, 2005). A single target gene often contains putative binding sites for multiple microRNAs that can bind cooperatively,allowing microRNAs to form complex regulatory control networks. microRNAs play key regulatory roles in control of haematopoiesis, developmental timing, cell differentiation, apoptosis, cell proliferation and organ development as well as in cancer, infectious disease, genetic disorders (Lin et al, 2006) and even heart disease (van Rooij et al, 2006).

microRNA biosynthesis and function

MicroRNAs are transcribed in a RNA Polymerase II-dependent manner as large polyadenylated pri-microRNAs. MicroRNAs are transcribed in a RNA Polymerase II-dependent manner as large polyadenylated pri-microRNAs. RNAPII catalyzes the transcription of DNA to synthesize precursors of mRNA and most snRNA and microRNA Yang CGFR 16:397, 2005

Pri-microRNAs are cleaved within the nucleus by Drosha, an RNaseIII-type nuclease, to form pre-microRNA 60–70 nucleotide hairpin structures. BJH 137,

Drosha requires the cofactor DiGeorge syndrome critical region 8 gene (DGCR8) in humans (Yeom et al, 2006). BJH 137,

The pre-microRNAs are exported from the nucleus to the cytoplasm by Exportin5 (Zeng, 2006). BJH 137,

The cytoplasmic pre-microRNA is further cleaved to form an asymmetric duplex intermediate (microRNA: microRNA*) by Dicer, another RNaseIII- type enzyme. Similar to Drosha, cofactors such as TRBP and PACT (in humans) are necessary for Dicer activity (Lee et al, 2006). The cytoplasmic pre-microRNA is further cleaved to form an asymmetric duplex intermediate (microRNA: microRNA*) by Dicer, another RNaseIII- type enzyme. Similar to Drosha, cofactors such as TRBP and PACT (in humans) are necessary for Dicer activity (Lee et al, 2006).

microRNA:microRNA* duplex is in turn loaded into the miRNA- induced silencing complex miRISC

The consequence of miRISC-loaded microRNAs is largely dependent upon the degree of complimentarity between the microRNA and its target gene. The consequence of miRISC-loaded microRNAs is largely dependent upon the degree of complimentarity between the microRNA and its target gene. It leads to either degradation of mRNA or blockage of translation without degradation. It leads to either degradation of mRNA or blockage of translation without degradation.

Cell, Vol. 116, 281–297, January 23, 2004 The choice of posttranscriptional mechanisms is not determined by whether the small silencing RNA originated an siRNA or a miRNA but instead is determined by the identity of the target.

Aberrant expression of microRNA The majority of human microRNAs are located at cancer-associated genomic regions (Calin et al, 2004a). microRNA expression profiling can distinguish cancers according to diagnosis and developmental stage of the tumour to a greater degree of accuracy than traditional gene expression analysis (Lu et al, 2005). MicroRNAs play a direct role in oncogenesis as they can function as both oncogenes (e.g. MIRN155 and members of MIRN17–92 cluster) and tumour suppressor molecules [e.g. MIRN15A (miR-15a) and MIRN16-1 (miR-16-1)]. Aberrant expression of specific microRNAs has now been associated with many types of cancer including solid and haematopoietic tumours.

microRNA expression in leukaemia Expression levels of MIRN15A and MIRN16-1, encoded within the 13q14 region, were downregulated in 75% of CLL cases that harboured this chromosomal abnormality. These microRNAs were subsequently shown to target BCL2 and to induce apoptosis in vitro, suggesting they have tumour-suppressor role in CLL (Cimmino et al, 2005). MIRN16-1 negatively regulates cellular growth and cell cycle progression (Linsley et al, 2007). A follow-up study (Calin et al, 2005) of 94 CLL cases, defined a prognostically significant 13-gene microRNA signature by expression profiling. Moreover two of the CLL patients were found to have germline mutations in the MIRN16-1/MIRN15A precursor sequence that resulted in reduced expression levels of these microRNAs both in vitro and in vivo.

RNAi gene therapy application Viral infections: - HIV - Hep B - Hep C - RSV Cancer Neurodegenerative disorders: - Spinocerebellar Ataxias - Huntington disease - alzheimer disease Ocular disorders (Macular degeneration) Stem cell biology and therapy

ASH education book 2007