DOI:10.1158/0008-5472.

CAN-05-0298

MicroRNA Biogenesis and Cancer

Richard I. Gregory and Ramin Shiekhattar

Cancer Res 2005;65:3509-3512. Published online May 2, 2005.

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 DOI:10.1158/0008-5472.CAN-05-0298

Review

MicroRNA Biogenesis and Cancer

Richard I. Gregory and Ramin Shiekhattar
Gene Expression and Regulation Program, and Molecular and Cellular Oncogenesis Program, Wistar Institute, Philadelphia, Pennsylvania

Abstract this promoter, although able to direct polymerase IIdependent

MicroRNAs (miRNA) are a recently discovered family of short transcription, lacks all typical promoter elements normally
nonprotein-coding RNAs that negatively regulate gene required for transcription initiation. The significance and genera-
expression. Recent studies of miRNAs highlight a requirement lity to miRNA gene promoters of this observation is currently
for cell viability. Posttranscriptional silencing of target genes unknown. A subset of human miRNA genes are located within
by miRNAs occurs either by targeting specific cleavage of introns of pre-mRNAs. Because these miRNAs have the same
orientation as pre-mRNAs, it is likely that they are processed from
homologous mRNAs, or by targeting specific inhibition of
the introns and one would therefore expect the tissue-specific
protein synthesis. We recently identified a multisubunit
expression profile of the miRNA to correlate with that of the
protein complex termed Microprocessor that is necessary
mRNA. The remaining miRNAs are clustered in the genome
and sufficient for processing miRNA precursor RNAs. Micro-
predicting a long transcript encompassing several coordinately
processor contains Drosha, an RNase III endonuclease, and
expressed miRNAs. Irrespective of how different miRNAs originate,
DGCR8, a gene deleted in DiGeorge syndrome. We consider
the pri-miRNA transcripts are predicted to form specific hairpin-
recent findings that link miRNA perturbation to cancer.
shaped secondary structures and are processed to yield a mature
(Cancer Res 2005; 65(9): 3509-12)
22-nucleotide miRNA.
Previously, it was established that processing of pri-miRNAs
The discovery that small (f22 nucleotides), noncoding, in the nucleus is mediated by Drosha, a RNase III endonuclease
double-stranded RNA molecules can mediate the expression of (5). Drosha asymmetrically cleaves both strands of the hairpin
target genes with complementary sequence led to the identifica- stem at sites near the base of the primary stem loop thus relea-
tion of a large family of evolutionary conserved, regulatory RNAs, sing a 60- to 70-nucleotide pre-miRNA that has a 5V phosphate
dubbed microRNAs (miRNAs; refs. 1, 2). In mammals, hundreds and a 2-nucleotide 3V overhang. Specific RNA cleavage by Drosha
of miRNAs have now been identified, some of which are expressed predetermines the mature miRNA sequence and provides the
in a tissue-specific and developmental stagespecific manner. For substrate for subsequent processing events. The pre-miRNAs are
the few miRNAs whose function has been uncovered, they are transported to the cytoplasm by Exportin-5, in a Ran GTP-
important regulators of various aspects of developmental control dependent manner. The interaction of Exportin-5 with the pre-
in both plants and animals (1, 2). In the few years since the miRNA intermediate and its subsequent transport requires
inception of this regulatory RNA phenomenon, much progress correctly processed pre-miRNAs with hallmarks of Drosha-
has been made towards an understanding of the mechanisms by mediated cleavage and specific hairpin secondary structure
which this occurs and the identification of cellular machinery (6, 7). Once in the cytoplasm, a second RNase III endonuclease,
involved in RNA-mediated silencing (1, 2). In this review, we Dicer, acts on the pre-miRNA. It is thought that the PAZ domain
focus on recent discoveries related to the miRNA biogenesis of Dicer recognizes the 2-nucleotide 3V overhang at the base of
pathway and discuss the implications for human diseases the stem loop of the pre-miRNA. Dicer cleaves double stranded
including cancer. RNA 22-nucleotides from the end of the substrate (8). In the case
of pre-miRNA, one end of the miRNA has already been pre-
determined by Drosha cleavage site selection. Therefore, a
MicroRNA Transcription and Stepwise Maturation subsequent cleavage by Dicer releases a 22-nucleotide mature
The majority of the characterized miRNA genes are intergenic double-stranded miRNA with 5Vphosphates and a 2-nucleotide 3V
or oriented antisense to neighboring genes and are therefore overhangs. One strand of the miRNA duplex is subsequently
suspected to be transcribed as independent units. The primary incorporated into an effector complex termed RNA-induced
transcripts (pri-miRNA) are generated by polymerase II and recent silencing complex that mediates target gene expression (reviewed
data indicate that pri-miRNAs possess a 5V7-methyl guanosine cap in detail in ref. 2).
and are polyadenylated (3, 4). Probably the best-characterized
human pri-miRNA is that of miR-23af27af24-2, a 2.2-kb
transcript containing three miRNAs. Pri-miR-23af27af24-2 is 5V Processing of Primary MicroRNAs by the
capped and polyadenylated f1.8 kb downstream of the 3Vend of Microprocessor Complex
miR-24-2. A minimal (f600 bp) polymerase IIdependent As discussed, Drosha plays an essential role in the genesis of
promoter was identified at this miRNA gene (3). Interestingly, miRNAs; however, it was not known whether other Drosha-
associated components contribute to the processing of pri-
miRNAs. Recently, four groups independently investigated the
potential involvement of other proteins in pri-miRNA processing
Requests for reprints: Ramin Shiekhattar, Wistar Institute, 3601 Spruce Street, (912). To address the question of whether Drosha-associated
Philadelphia, PA 19104. Phone: 215-898-3896; Fax: 215-898-3868; E-mail:
[email protected]. components contribute miRNA biogenesis, we biochemically
I2005 American Association for Cancer Research. purified Drosha from human cells by affinity chromatography

www.aacrjournals.org 3509 Cancer Res 2005; 65: (9). May 1, 2005

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Copyright 2005 American Association for Cancer Research
 DOI:10.1158/0008-5472.CAN-05-0298
Cancer Research

and identified f20 Drosha-associated proteins by mass spectro- proteins were purified and assayed for pri-miRNA processing.
metric sequencing (9). Fractionation of the affinity eluate by a Neither recombinant Drosha nor DGCR8 showed any significant
gel filtration column revealed one complex (>2 MDa) containing miRNA processing activity. However, addition of the both recom-
most of the Drosha-associated proteins, and a smaller Drosha- binant proteins together reconstituted the miRNA processing
containing complex of f600 kDa (9). Previously, an in vitro assay activity to similar levels seen with native complex (9). Moreover,
was established to monitor pri-miRNA processing using in vitro Drosha protein itself displayed a nonspecific RNase activity
transcribed RNAs corresponding particular to pri-miRNAs (13). toward the substrate, which may provide insight into the
We cloned a miRNA gene cluster corresponding to miRNAs 17-18- mechanism of Microprocessor action by demonstrating the
19a-20-19b. After in vitro transcription, this fragment, containing requirement of DGCR8 in directing the specific cleavage of pri-
five miRNAs, generates a pri-miRNA of f800 nucleotides. Using miRNA by Drosha. As the DGCR8 protein contains two double-
this pri-miRNA as a substrate, we assayed the column fractions stranded RNA binding domains at its COOH terminus, it is
for processing activity and observed accumulation of a 63- tempting to speculate that the role of DGCR8 in the Micropro-
nucleotide pre-miRNA that coeluted with the smaller (f600 kDa) cessor may be to recognize pri-miRNAs and/or to orient the
Drosha-containing complex (9). We determined the polypeptide catalytic RNase III core of Drosha to ensure correct cleavage site
composition of this complex by mass spectrometric sequencing selection at the stem of the hairpin RNA structure. Furthermore,
and found two proteins: Drosha and the double-stranded RNA- DGCR8 has an NH2-terminal WW domain known to interact
binding protein DGCR8 (9). We confirmed this interaction by with proline-rich peptides. The WW domain of DGCR8 is most
purification of Drosha as a DGCR8-associated protein and showed likely the interacting surface with the proline-rich NH2-terminal
pri-miRNA processing by the complex. Concomitantly, the domain of Drosha. This possibility requires further exploration
Hannon group was investigating the association of a candidate based on the observation of Han et al., where it seemed that
protein (CG1800) that was previously shown to potentially the proline-rich domain of Drosha is dispensable for interaction
interact with Drosha based on yeast two hybrid data (10). with DGCR8 (11); however, it should be noted that interpretation
Consistent with our discovery in human cells, when extract from of this result may be complicated by the indication that a single
Drosophila S2 cells was fractionated by gel filtration, a peak of Microprocessor complex is formed by multiple molecules of
miRNA processing (of pri-bantam RNA) was observed in frac- Drosha and/or DGCR8 (11).
tions corresponding to a protein complex of f600 kDa, that Of particular interest is the fact that DGCR8 was originally
comprised Drosha and the Drosophila orthologue of DGCR8, a identified as a gene that maps to the chromosomal region 22q11.2,
protein they named Pasha (partner of Drosha; ref. 10). This a region whose monoallelic deletion accounts for >90% of patients
complex, comprising Pasha/DGCR8 and Drosha, was named the with DiGeorge syndrome, the most common human genetic
Microprocessor complex (9, 10). A third group investigated the deletion syndrome that effects around 1 in 3,000 live births (14).
molecular mechanism of pri-miRNA processing by Drosha, The clinical manifestations of the disease are highly variable, with
when they fractionated nuclear extract from human cells and 75% of patients displaying congenital heart defects. Other
assayed it for pri-let-7-a-1 miRNA processing activity, a distinct common features include characteristic facial appearance, immu-
peak of activity was detected in fractions corresponding to a nodeficiency from thymic hypoplasia, and developmental and
molecular mass of >700 kDa (11). This peak of activity shifted behavioral problems, including schizophrenia and obsessive-
to f650 kDa after the nuclear extract was treated with RNase compulsive disorder in adulthood (15). Despite its relatively high
A before gel filtration. This complex most likely corresponds incidence, the fact that most cases of DiGeorge syndrome are
to Microprocessor, and indicates that the integrity of this complex caused by haploinsufficiency of a typical deleted region (f1.5 Mb)
is mediated by protein-protein interactions and does not depend that encompasses around 30 genes has made the task of
on RNA (11). Together, these three studies provide very strong identifying the particular gene(s) that underlie the pathogenesis
support for the existence of Microprocessor, a Drosha-containing a challenge. Given the emerging role of miRNAs in regulating
complex of around f600 kDa that is conserved from flies to diverse aspects of development, it is tantalizing to hypothesize
human (see Fig. 1; refs. 911). that haploinsufficiency of DGCR8; thus, defects in miRNA
To assess the role of Drosha-DGCR8 complex in initiation of biogenesis may contribute to the widespread developmental
miRNA processing in vivo, we used RNA interference to deplete abnormalities affecting DiGeorge syndrome patients. In this
DGCR8 and Drosha in human cells. Small interfering RNA against respect, we suggest that DGCR8 is strong candidate gene for
Drosha and DGCR8 invoked a pronounced decrease in mature DiGeorge syndrome and are currently investigating the potential
miRNA levels (9). Consistent with Microprocessors role in role of miRNA perturbation in the genesis and development of
processing pri-miRNAs, we also detected an accumulation of DiGeorge syndrome.
pri-miRNA following knock down of Drosha or DGCR8 (9). In
accordance with our findings, genetic studies in Caenorhabditis
elegans using mutant Drosha and mutant Pasha worms (10), Cancer Connections
together with RNA interferencemediated knockdown of these Insights leading to the understanding of miRNA biogenesis may
proteins in Drosophila cells (10, 12), C. elegans (10), and human affect cancer in at least two ways. First, emerging data indicate that
cells (1012), confirmed an obligatory role for these proteins in dysregulation of miRNAs is associated with certain types of cancer
processing pri-miRNA to pre-miRNA. Although the aforemen- (1620). Second, exploitation of the therapeutic potential of RNA
tioned data show that both Drosha and DGCR8 are necessary for interference may be achieved through the investigation of how
pri-miRNA processing, the most compelling evidence for the endogenous miRNA and produced and exert their gene regulatory
sufficiency of these proteins in mediating miRNA biogenesis was function (21, 22). Chronic lymphocytic leukemia (CLL), the most
provided by our ability to reconstitute Microprocessor activity common form of adult leukemia, can be attributed to a deletion at
using recombinant proteins (9). Recombinant Drosha and DGCR8 13q14 in >50% of cases. Additionally, various other cancers

Cancer Res 2005; 65: (9). May 1, 2005 3510 www.aacrjournals.org

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Copyright 2005 American Association for Cancer Research
 DOI:10.1158/0008-5472.CAN-05-0298
MicroRNA Biogenesis and Cancer

Figure 1. Model of miRNA biogenesis. Long primary transcripts (pri-miRNAs) containing one to several miRNAs are generated by polymerase II and are 5Vcapped
and polyadenylated. The recently identified Microprocessor complex, comprising Drosha (RNase III endonuclease) and DGCR8 (a double-stranded RNA binding
protein) recognize the distinct hairpin secondary structure of the pri-miRNA and specifically cleave at the base of the stem loop releasing a 60- to 70-nucleotide
pre-miRNA intermediate enabling Exportin 5mediated cytoplasmic export where Dicer, a second RNase III endonuclease, cleaves 22-nucleotide from the Drosha
cleavage site to yield the mature miRNA. Dysregulation of this processing leading to perturbation in miRNA genesis may have oncogenic consequences.

(including mantle cell lymphoma, multiple myeloma, and prostate that miRNAs are frequently located at genomic regions involved in
cancers) have been linked to varying degrees with 13q14 deletions. cancers, and their gene regulatory function, that miRNAs may act as
Despite considerable effort, none of the known genes located in the both tumor suppressors and oncogenes (20). In addition, although
deleted region have been shown to lead to CLL. Two miRNA genes, the function of the larger (>2 MDa) Drosha-containing complex we
miR-15 and miR-16, map to this deleted region and it was found that purified is currently unknown, attention should be drawn to our
in 68% of CLL patients and the majority of prostate cancer cell lines identification of Ewings sarcoma gene product (EWS) as one
tested, both miRNA genes are deleted or down-regulated. Perhaps component (9). Chromosomal translocations of the EWS gene that
even more provocative is the observation that in a subset of CLL encodes a putative RNA binding protein, are known to cause Ewing
samples, accumulation of the (f70 nucleotides) pre-miR-15 sarcoma as well as neuroectodermal and various other tumors (9).
intermediate was detected by Northern blotting, pointing to a Future investigations will undoubtedly reveal additional links
potential deficiency in miR-15 processing (16). Down-regulation of between mechanisms of miRNA-mediated gene silencing and
miR-143 and miR-145 was observed in colorectal neoplasia (17), and human diseases including cancer.
expression of the miRNA let-7 is frequently reduced in lung cancers,
a feature that is associated with poor prognosis (18). Increased Acknowledgments
expression of the precursor of miR-155 was detected in pediatric
Received 1/28/2005; revised 2/9/2005; accepted 2/11/2005.
Burkitt lymphoma (19). In fact, it has been speculated based on Grant support: Jane Coffin Childs Foundation Fund for Medical Research
cancer-associated alterations in miRNA expression, the observation postdoctoral fellowship (R.I. Gregory).

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