Yoko Fukuda Hiroaki Kawasaki and Kazunari Taira Summary

MicroRNAs (miRNAs) are a class of noncoding RNAs that regulate gene expression by single-stranded RNAs of 18 to 25 nucleotides in length. Hundreds of miRNAs have been found in animals and plants, some of which play important roles in development or differentiation. Increasing attention has thus been paid to their biogenesis and regulation mechanisms and the identification of target genes. We are constructing a comprehensive expression vector library containing predicted human miRNAs. miRNA expression vectors containing human RNA polymerase II or III promoters, and utilizing a flexible vector system, can be useful for functional analysis.

Key Words: MicroRNA; CMV promoter; U6 promoter. 1. Introduction

MicroRNAs (miRNAs) are a class of tiny noncoding RNAs (1-3) that regulate the expression of target genes by translational repression or mRNA cleavage (4,5). Since the first discovery of lin-4 and let-7in Caenorhabditis elegans (6,7), hundreds of miRNA have been found in animals and plants, and their functions are continuously being unveiled. At present, mammalian miRNAs are associated with hematopoietic differentiation (8), adipocyte differentiation (9), insulin secretion (10), and some other important mechanisms.

The primary transcripts of miRNA genes form characteristic stem-loop structures (11), which are initially processed by RNase III Drosha (12); the resultant pre-miRNAs are then exported to the cytoplasm. Mature miRNAs are then generated after processing of pre-miRNAs by RNase III Dicer (12) and incorporated into micro-ribonucleoproteins (miRNPs) (13). In miRNPs, miRNAs act as the "guide" sequences that recognize complementary mRNA sequences.

From: Methods in Molecular Biology, vol. 338: Gene Mapping, Discovery, and Expression: Methods and Protocols Edited by: M. Bina © Humana Press Inc., Totowa, NJ

Mature or precursor sequences of predicted miRNAs and their genomic positions are available in the public miRNA Registry database (http://www.sanger. ac.uk /Software/Rfam/mirna/index.shtml). For comprehensive analysis of the functions and molecular mechanisms of human miRNAs, we have constructed an expression vector library of human miRNAs from this database.

The transcription of miRNAs is not yet fully understood. They were first believed to be transcribed by RNA polymerase III (pol III); however, some miRNAs are probably transcribed by RNA pol II (14). Although mature miRNAs have a very short sequence of 18 to 25 nucleotides, their pri-miRNAs are relatively long, sometimes over several kilobases, and have four or more uridine residues (11). Because these observations are characteristic of pol II transcripts, unlike most small RNAs that are RNA pol III transcripts, a considerable number of miRNAs are thought to be transcribed by RNA pol II (4). We have constructed two types of miRNA expression vectors. The system mainly introduced here uses a pol II promoter; miRNA genes are under the control of a cytomegalovirus (CMV; pol II) promoter and can easily be transferred to other expression vectors by the TOPO Gateway system. Detailed analysis of time- and tissue-specific expression of miRNAs can be conducted using this flexible vector system. The other system uses a U6 (pol III) promoter, is suitable for expressing short-length RNA, and allows high expression levels of desired sequences in the transfected cells. Since about 30-bp flanking regions of predicted stem-loop sequences can be necessary for maturation of miRNAs when they are constructed in expression vectors (8), all miRNA expression vectors containing predicted miRNA precursor sequences have been constructed with additional 30 to 40-bp genomic sequences.

This chapter describes the construction of miRNA expression vectors driven by human RNA pol II and III promoters (Figs. 1-3).

Diabetes 2

Diabetes 2

Diabetes is a disease that affects the way your body uses food. Normally, your body converts sugars, starches and other foods into a form of sugar called glucose. Your body uses glucose for fuel. The cells receive the glucose through the bloodstream. They then use insulin a hormone made by the pancreas to absorb the glucose, convert it into energy, and either use it or store it for later use. Learn more...

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