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microRNAs and Apoptosis

microRNAs and Apoptosis

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An essay for the 2011 Undergraduate Awards (Ireland) Competition by None.
An essay for the 2011 Undergraduate Awards (Ireland) Competition by None.

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Published by: Undergraduate Awards on Aug 31, 2012
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10/27/2013

 
mi croRNAs and Apoptosis
Introduction
microRNAs (miRNAs) are non-protein-encoding RNA molecules that function inthe regulation of gene expression. miRNAs negatively regulate gene expression bycausing target mRNA molecules to undergo mRNA cleavage or translationalrepression. There is increasing evidence that miRNAs play a significant role in theregulation of apoptosis. We will first examine miRNA biogenesis and miRNAregulation of gene expression. We will then explore the experimental evidence thatsupports the role of miRNAs in both the regulation and deregulation of apoptosis. Wewill conclude with a discussion of the future prospects for the use of miRNAs intherapeutic applications.
m icroRNAs
miRNAs are highly conserved, single-stranded RNA molecules consisting of ~19-25 ribonucleotides (1,2). miRNA genes comprise ~1% of the human genome and arethought to regulate the expression of up to one third of human genes (3,4). ManymiRNA genes are located in regions > 1 kb away from known genes which suggeststhat they can be transcribed as independent units (3). A small number of miRNAgenes have also been found in the introns of pre-mRNAs (5). However, ~50% of miRNA genes are located in clusters, indicating that several miRNA genes can betranscribed as a ‘multi-cistronic primary transcript’ (2,3)
(I ) microRNA biogenesis
miRNA biosynthesis occurs in numerous steps (Fig.1). The miRNA gene istranscribed by RNA polymerase II to form a primary miRNA (pri-miRNA) transcriptthat contains a 5’ 7-methylguanosine cap and a 3’ poly-A-tail (2). It has a hairpinstructure containing a stem loop and flanking segments (2). The pri-miRNA transcriptthen undergoes maturation which occurs in three steps.1
 
In step one, known as Cropping,
 
the pri-miRNA transcript is bound by themicroprocessor complex which consists of Drosha and its cofactor, Di Georgesyndrome critical region gene 8 (DGCR8) protein (3). The double-stranded RNA- binding domains of Drosha and DGCR8 protein recognise the double-stranded stemstructure of the miRNA by measuring the stem length since the cleavage site is positioned ~ 22 nucleotides from the terminal loop (3). The RNase III domains of Drosha catalyse the cleavage of both strands of the pri-miRNA transcript to form astem loop precursor miRNA (pre-miRNA) (1). The staggered cleavage causes the base of the pre-miRNA stem loop to have a free terminal 5’ phosphate and a 2nucleotide 3’ overhang (5). When the miRNA gene is located within the intron of a protein-encoding gene, Drosha can release the miRNA before mRNA splicing occursso that both miRNA and mRNA can be generated simultaneously (2). Thismechanism allows miRNAs to directly regulate the expression of target intronic pre-mRNAs.In step two, the nuclear transport receptor, Exportin-5, binds the pre-miRNA in thenuclear membrane and cytoplasmic Ran GTPase-activating protein hydrolyses Ran-GTP to Ran-GDP to release the pre-miRNA into the cytoplasm (1,2). In step three,known as
 
Dicing, Dicer, an ATP-dependent RNase III, recognises the specific 3’overhang created by Drosha. With the help of 
trans-activator RNA (tar) - binding  protein
(TRBP), Dicer cleaves both strands of the pre-miRNA at the basal loop of thehairpin structure to form a duplex molecule of ~22 nucleotides (1,3). Therefore, the precision of the cleavage made by the microprocessor complex is essential for correctdicing because a change in the pre-miRNA cleavage site of Drosha would change thecleavage site of Dicer, resulting in the formation of different 3’ and 5’ ends in themature miRNA molecule (2).The duplex molecule contains a mature miRNA strand (guide strand) and afragment called miRNA* (passenger strand) (1,2). The arms of the stem-loopstructure are imperfectly paired causing one strand to be less stably paired at its 5’end, which is the mature miRNA strand (2). As a result, the 5’ terminus of the guidestrand has lower thermodynamic stability than that of the passenger strand (2). ThemiRNA:miRNA* molecule contains mismatches and bulges that allow the guidestrand to be bound by the large protein effector complex, the RNA-induced silencingcomplex (RISC) via the Argonaute 2 protein (Ago2) (1,4). The Argonaute proteinfamily consists of ~100 kDa proteins which contain ‘piwi-argonaute-zwille’ (PAZ)2
 
and PIWI structural domains (6). The PAZ domain of Ago2 contains a binding pocketfor the 2 nucleotide 3’ overhang created by Drosha and a DDH catalytic triad thatcatalyses endonucleolytic cleavage (6). The unwinding of the miRNA:miRNA*duplex activates RISC and the miRNA* strand is cleaved by Ago2 and then degraded(1,7). The guide strand directs the RISC complex to its target mRNA molecule toallow the miRNA prevent mRNA translation (1).3

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