The International Journal of Biochemistry & Cell Biology
mir-17-92, a cluster of miRNAs in the midst of the cancer network
Introduction
Data from comparative genomic studies have long hinted at the functional importance of the non-protein coding regions of the genome, whose proportion increases as a function of genomic complexity (Mattick and Makunin, 2006). Extensive transcription has been observed from many of these regions, giving rise to numerous non-coding RNAs (ncRNAs) that regulate diverse developmental and physiological processes (Kapranov et al., 2007). We are only beginning to understand the realm of ncRNA functions, yet preliminary studies so far have revealed unexpected complexity and richness in their expression patterns, genomic structures, and biological activities. In recent years, microRNAs (miRNAs) have emerged as a class of novel small ncRNAs with potent capacity for gene regulation at the post-transcriptional level (Zamore and Haley, 2005, He and Hannon, 2004, Bartel, 2009, Ambros, 2004). First identified as novel ncRNAs essential for larval developmental timing in C. elegans (Wightman et al., 1993, Lee et al., 1993), thousands of such small RNAs have been and are still being discovered in almost all organisms, ranging from viruses and single-celled green algae Chlamydomonas (Zhao et al., 2007, Molnar et al., 2007) to complex mammalian species (Lee and Ambros, 2001, Lau et al., 2001, Lagos-Quintana et al., 2001). Upon maturation, miRNAs recognize specific mRNA targets through imperfect sequence complementarity and largely dampen their expression at the post-transcriptional level. Given the ability of miRNAs to down-regulate many mRNA targets, this level of gene regulation may provide robustness in a diverse range of biological processes, due to the simultaneous regulation of many components of the signaling network.
Genes encoding miRNAs are transcribed as long primary transcripts (pri-miRNAs) that contain a stem-loop hairpin structure(s) (Lee et al., 2002). Pri-miRNAs are sequentially processed by two RNaseIII enzymes, Drosha and Dicer, to yield mature miRNA duplexes ranging from 18 to 24 nucleotides in length (Lee et al., 2003, Hutvagner and Zamore, 2002). One strand of the mature duplex is then incorporated into the effector complex, the RNAi-induced silencing complex (RISC), to mediate the post-transcriptional silencing of specific mRNAs (Hutvagner and Zamore, 2002, Mourelatos et al., 2002). The recognition of the miRNA targets often involves imperfect base pairing (Wightman et al., 1993, Lee et al., 1993), which allows miRNA to potentially regulate a large number of protein coding genes (Lewis et al., 2003, Lewis et al., 2005, Krek et al., 2005, Grimson et al., 2007). Interestingly, multiple miRNAs can be produced within a single pri-miRNA transcript, each of which can act independently. This polycistronic structure of miRNA cluster genes sets them apart from most protein coding genes in animals and bestows upon them a unique capacity and specificity for widespread gene regulation in the complex molecular networks for development and disease.
Despite the small size of the miRNA molecules, they exhibit enormous capacity for gene regulation, mostly due to the nature of imperfect base pairing required for target recognition (Lewis et al., 2003, Lewis et al., 2005, Krek et al., 2005, Grimson et al., 2007). Therefore, each miRNA is theoretically capable of regulating hundreds of mRNAs within a cell type and in a context-dependent manner. Initially, a small number of miRNA targets were identified based on classic invertebrate genetics studies (Johnston and Hobert, 2003, Brennecke et al., 2003, Wightman et al., 1993, Lee et al., 1993). Examination of these validated target sites combined with biochemical studies revealed that the 5′ end of a miRNA, designated as the seed sequence, plays a critical role in target recognition and post-transcriptional repression (Lewis et al., 2005, Grimson et al., 2007, Doench and Sharp, 2004). The miRNA seed complementarity was exploited to develop computational approaches for target prediction, and was later demonstrated experimentally to be the major molecular basis for miRNA target recognition in cell culture systems (Selbach et al., 2008, Baek et al., 2008). Exceptions to this generalization still exist (Didiano and Hobert, 2006, Wightman et al., 1993). For example, in C. elegans, a 6–8-base-pair perfect seed pairing is not always a reliable predictor for the lsy-6 miRNA interaction with its targets, because it has been shown that G/U base pairing is tolerated in the seed region. It is likely that a variety of mechanisms may act together to regulate the specificity of the miRNA:target interaction (Didiano and Hobert, 2006).
The exact molecular basis underlying miRNA-mediated gene silencing is not entirely clear. Biochemical studies using different reporter systems and endogenous miRNAs have yielded several distinct models, possibly reflecting the limitation of the in vitro reporter systems tested and/or the complex nature of miRNA-mediated gene silencing (Liu et al., 2005, Kiriakidou et al., 2007, Eulalio et al., 2008, Chendrimada et al., 2007, Bhattacharyya et al., 2006). In all these models, miRNAs, upon maturation, are incorporated into the RISC (Hutvagner and Zamore, 2002), which mediates degradation of specific mRNAs (Eulalio et al., 2008, Giraldez et al., 2006, Bagga et al., 2005) and/or represses their translation (Chendrimada et al., 2007, Kiriakidou et al., 2007, Pillai et al., 2005). The key components of the RISC are the Argonaute (Ago) proteins, which, through their interactions with the P-body components, mediate the formation of P-bodies that contain miRNAs and their targets (Eulalio et al., 2008, Liu et al., 2005, Sen and Blau, 2005). Within the P-body, miRNA targets are sequestered from the translational machinery and are subjected to mRNA degradation, at least partially, through deadenylation (Giraldez et al., 2006). In several other studies, Ago proteins are suggested to mediate translational repression through various mechanisms (Filipowicz et al., 2008). Although there is debate about the exact mechanism of miRNA-mediated repression, these findings imply that both mRNA destabilization and translational repression contribute to miRNA-mediated gene silencing. This is consistent with recent findings using genome-wide quantitative mass spectrometry to characterize the impact of miRNAs on the proteome in a cell culture system (Baek et al., 2008, Selbach et al., 2008). It is likely that multiple molecular pathways, either individually or collectively, mediate the biological effects of miRNAs in a context-dependent manner.
miRNAs were initially studied as key regulators for normal development, as mutations, either in the founding members of the miRNA family or in the key components of the miRNA biogenesis pathway, give rise to pronounced developmental defects in nearly all model organisms. Even though all miRNAs share significant similarities in their gene structures, biogenesis, and effector machineries, their expression patterns and biological functions vary tremendously. This unique mechanism of post-transcriptional gene silencing, along with transcription regulation, translation control, and post-translation modification, are major gene regulatory mechanisms in diverse developmental and physiological processes.
Shortly after the identification of miRNAs in mammalian species, it was recognized that miRNAs may not solely regulate developmental processes. Initial studies indicated that genomic alterations and expression dysregulation of miRNAs are frequently associated with various human cancers (Calin et al., 2004a, Calin et al., 2004b, Iorio et al., 2005, Lu et al., 2005). These early findings prompted a number of subsequent studies to explore the functional connections between miRNAs and malignant transformation in a variety of cancer types. Two major approaches were undertaken. One approach aimed to identify candidate oncogenic and tumor suppressor miRNAs for the transformation of a specific cell type. This often involved performing expression studies to compare miRNA profiles between tumor and normal tissues. Under this rationale, mir-17-92 was identified as a potential oncogene due to its genomic amplification and elevated expression in multiple hematopoietic malignancies, including diffuse large B-cell lymphomas (DLBCLs), mantle cell lymphomas, and Burkitt's lymphomas (Hayashita et al., 2005). Previous cancer genomic studies on DNA copy number alteration and insertional mutagenesis screens in mice also provided insight into candidate oncogenic and tumor suppressor miRNAs that were not fully recognized before. For example, the oncogenic potential for mir-155 and mir-106a-92 in lymphomagenesis were identified through this approach (Kluiver et al., 2005, Uren et al., 2008). The other rationale aims to explore the molecular crosstalk between miRNAs and well-characterized cancer pathways. From expression studies, miRNA functional screens, and computational analyses, a handful of candidate miRNAs have emerged as key components of the oncogenic and tumor suppressor network. For example, it is through extensive expression studies that mir-34 was first identified as a p53 transcriptional target, mediating its tumor suppressor effects (Raver-Shapira et al., 2007, He et al., 2007, Chang et al., 2007); mir-372 and mir-373 were identified as potential oncogenes for testicular tumorigenesis out of a functional screen using a miRNA over-expression library (Voorhoeve et al., 2006); and finally, bioinformatic prediction of let-7 miRNA targets led us to explore its roles in post-transcriptional repression on key oncogenes Ras and HMGA2 (Johnson et al., 2005, Park et al., 2007, Mayr et al., 2007). Altogether, these lines of investigation led to a surge of interest in the functional studies of miRNAs in the oncogenic and tumor suppressor network, an area that has been largely unexplored until recently.
Section snippets
The unique gene structure of mir-17-92
One of the best-characterized oncogenic miRNAs is mir-17-92, a polycistronic miRNA cluster also designated as oncomir-1 (He et al., 2005). The precursor transcript derived from the mir-17-92 gene contains six tandem stem-loop hairpin structures that ultimately yield six mature miRNAs: miR-17, miR-18a, miR-19a, miR-20a, miR-19b-1, and miR-92-1 (Tanzer and Stadler, 2004). Recent high-throughput parallel sequencing efforts also discovered the rare species of miRNA star forms (miRNAs*) within this
Summary
Recent studies have revealed the unexpected abundance and complexity of ncRNAs transcribed in the mammalian genome, the functions of which are largely unexplored. The identification of ncRNA components in the cancer pathway reflects the necessity for complex gene regulation to achieve homeostasis and flexibility to maintain a normal developmental or physiological state. mir-17-92 is among the first miRNAs recognized as key components of the molecular network that impact tumorigenesis and tumor
Acknowledgements
We thank Dr. Pengcheng Bu and Ms. Mona Foth for critical reading of this manuscript and for helpful discussions. L.H. is a fellow of the Searle Foundation and is supported by the K99 award from the NCI.
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