Analysis of microRNA Expression and Function - Caenorhabditis Elegans: Molecular Genetics and Development - page 238

reflect the transcriptional control of primary miRNAs production, and it cannot
provide information on post-transcriptional regulation or turnover.
Similarly, reporter gene fusion can also be used to analyze miRNA-mediated gene
regulation at the level of 3 0 UTRs (see below), which contain regulatory elements
such as miRNA-binding sites.
P. miRNA Promoter - Reporter Fusion
The first step in designing a miRNA promoter-reporter fusion construct is to
decide on the putative promoter sequence. Several factors should be taken into
consideration, including the intergenic, intragenic, or operonic location of miRNA
loci and the distance to the next upstream transcript, as well as the phylogenic
conservation of promoter sequences and the presence of known transcription fac-
tor-binding sites. Determination of the 5 0 end of the primary miRNA by 5 0 RACE
(see Section III) would help design the promoter-reporter fusion construct and it is
debatable whether the primary miRNA sequence upstream of the mature miRNA
should be kept in the construct. Previous studies have shown that fragments between
a few hundred basepairs to 2 kb in length upstream of the mature miRNA or known
pre-miRNA hairpin [miRBase ( Griffiths-Jones et al., 2008 )] contain sufficient
information to faithfully recapitulate the pattern of expression of endogenous
miRNAs. For example, we have used a 0.5 kb fragment upstream of lin-4 and a
2.2 kb fragment upstream of mir-84 as the promoters ( Esquela-Kerscher et al., 2005;
Johnson et al., 2005 ). Martinez et al. (2008) have used the intergenic genomic
sequences upstream of miRNA genes from 300 bp to 2 kb as the promoters in
their genome-scale analysis ( Martinez et al., 2008 ). Putative promoter regions
located in vast intergenic regions that lack homology across species or known
transcription factor binding sites may thus be safely limited to approximately 2 kb
in most cases.
The second step is to choose the reporter gene and the vector backbone. Andrew
Fire ' s laboratory has developed a large array of vectors and reporter gene derivatives,
using gfp or lacZ as the reporter, on the backbone of a pUC19 plasmid for fusion gene
expression in C. elegans (see Links). The cloning strategies described below are
adapted from several common cloning techniques for reporter gene fusions in
C. elegans ( Boulin et al., 2006 ). Due to the relatively short length of miRNA
promoters we have chosen, we only discuss three strategies more suitable for short
DNA fragment cloning, including conventional cloning, PCR fusion ( Horton et al.,
1989 ), and Multisite Gateway cloning ( Hope et al., 2004 ).
1. Conventional Cloning
The most common strategy to construct a miRNA promoter-reporter fusion is
conventional restriction enzyme-based cloning, graphically summarized in Fig. 2 A.
Perform PCR to amplify the promoter region ( 300 bp to 2 kb upstream in length