Biomedical Engineering Reference
In-Depth Information
1 Biogenesis of miRNAs
The field of RNA biology has come a long way since the early days when RNA
was considered to be just a passive messenger, carrying genetic information from
DNA to proteins (Crick, 1970). Early evidence for RNA being actively involved
in the regulation of gene expression can be traced back to 1961, when it was
suggested that RNA molecules can inhibit the expression of operons by blocking
operator function through base pairing (Jacob andMonod, 1961). However, one
of the most exciting developments in the study of RNA took place about a decade
ago, when microRNAs (miRNAs) were discovered (Lee et al., 1993; Wightman
et al., 1993). The miRNA database (miRBase:
http://microrna.sanger.ac.uk/
sequences
/) currently lists around 3097 mammalian miRNAs, with 678 miRNAs
identified in the human genome (version 11, April 2008). The database has seen a
dramatic increase in the number of listed miRNAs in various systems (from 218
in 2002 to 6396 in 2008). While most miRNAs are transcribed by RNA polII (Cai
et al., 2004; Lee et al., 2004), some, such as the cluster of miRNAs on chromo-
some 19, can be transcribed by RNA polIII (Cai et al., 2004). Based on their
location in the genome, many miRNA genes are transcribed as polycistronic
transcripts, as they tend to be clustered together (Lee et al., 2004). The physical
location of miRNA genes can be in various types of transcriptional units: in the
introns of protein-coding and non-protein-coding genes and in the exons of non-
protein-coding genes (Rodriguez et al., 2004). It has also been reported that when
an miRNA gene is in the imprinted locus of the genome, its expression is parent
specific, e.g., mir-127 and mir-136 (Seitz et al., 2003).
The primary miRNA transcripts (pri-miRNAs) form short double-stranded
hairpin structures and are processed by endoribonucleases and their associated
proteins in the nucleus and cytoplasm. In the nucleus, the endoribonuclease
Drosha and its interacting protein DGCR8 (microprocessor complex) excise
the stem loop of approximately 70 nucleotides with a 2-nucleotide (nt) 3
0
over-
hang from the pri-miRNA transcript (Lee et al., 2002). However, the discovery
of a pre-miRNA/intron (mirtrons) suggests that there is at least one alternative
mechanism of miRNA biogenesis that does not require microprocessor activity
for its maturation. The mirtrons are products of debranched introns that can
fold to have a stem-loop structure and contain a 5
0
monophosphate and 3
0
2-nt
overhang (similar to microprocessor-produced pre-miRNAs) (Okamura et al.,
2007; Ruby et al., 2007). The stem loop processed by the microprocessor
complex (pre-miRNA) and processed mirtron is then exported out of the
nucleus through a RanGTP and Exportin-5 complex in a GTP-dependent
manner (Bohnsack et al., 2004; Lund et al., 2004; Yi et al., 2003). In cytoplasm,
the pre-miRNA stem loop is further processed by another endoribonuclease,
Dicer, which recognizes the 2-nt overhang on the 3
0
end of the pre-miRNA
generated by Drosha activity. The N-terminal PAZ and c-terminal dsRNA-
binding domains of Dicer are responsible for its interaction with pre-miRNA
(Zhang et al., 2004). After Dicer forms a complex with pre-miRNA, the two
