RISCy business: microRNAs, pathogenesis, and viruses

Ben Berkhout1 and Kuan-Teh Jeang2*

From the Acdemisch Medisch Centrum1

Meibergdreef 15; 1105 AZ Amsterdam, the Netherlands; and the Laboratory of Molecular Microbiology2, NIAID, NIH, Bethesda, Maryland, 20892, USA.

Running title: microRNAs and diseases *Correspondence: KT Jeang, Building 4, room 306, NIH, 9000 Rockville Pike, Bethesda,

MD, 20892, USA. Tel: 301 496 6680; Fax: 301 480 3686; Email: kj7e@nih.gov

It has long been recognized that proteins serve important regulatory and effector functions inside cells. However, the recent discovery that plants and animals have thousands of genes that encode non-protein-coding (nc) RNAs has opened a new vista on RNA-mediated biology. NcRNAs include rRNA, tRNA, small nuclear (sn) RNA, small nucleolar (sno) RNAs, micro (mi) RNAs, and some of the lesser-known RNAs such as vault RNAs, Y RNAs, rasi-RNAs and piRNAs [reviewed in (1)]. Emerging data now suggest that whereas 2% of the human genome encodes for protein-coding RNAs, 60 to 70% of our DNA is transcribed into ncRNAs (2), (3). Thus, the earlier view that ncRNAs are largely, if not exclusively, constituted by the relatively abundant rRNAs, tRNAs, snRNAs and snoRNAs moieties is likely an oversimplification. More sensitive analytical methods such as RT-PCR and DNA tiling-arrays have revealed that genomes of complex organisms are replete with numerous less abundant ncRNAs which can contribute a hitherto unrecognized regulatory dimension. We review here in a non-exhaustive fashion the current, emerging and continuously evolving understanding of roles played by one class of ncRNA, the

miRNAs, in cellular metabolism, pathogenesis and host-pathogen interaction. miRNA biogenesis and mechanisms of action. The first miRNA, lin-4, and its target mRNA, lin-14, were described in Caenorhabiditis elegans by Ambros, Ruvkun and colleagues in 1993 [(4), (5); reviewed in (6), (7)]. Subsequently, computational analysis of aligned regions between human, mouse and puffer fish genomes initially led to the prediction of ~255 discrete miRNAs in the homo sapiens genome (8). That early number has been quickly exceeded by the latest enumeration of 474 characterized human miRNAs in the Sanger miRBase sequence database (release 9.1). Later in silico estimates have posited ~1,000 or more human miRNAs (9), (10); some of these newer suggestions have been verified by direct cloning and RNA-primed, array-based Klenow enzyme (RAKE) assay (11). Currently, difficulties with the reliability of computer-based miRNA prediction reside with the absence of any single property sufficient for accurate determination and the realization that novel miRNAs, by definition, may contain characteristics not fully recognized by extant pattern-algorithms (12). Additionally, experimental

http://www.jbc.org/cgi/doi/10.1074/jbc.R700023200The latest version is at JBC Papers in Press. Published on July 12, 2007 as Manuscript R700023200

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confirmation (e.g. direct cloning) of miRNAs remains challenging because expression profiles of ncRNAs can be constrained temporally, spatially and in a tissue-specific fashion. Moreover, the expression levels of individual miRNAs can vary by three orders of magnitude (13). Indeed, in a recent extensive miRNA cloning attempt to identify novel miRNAs, the investigators reported that 76% of their new miRNAs were cloned only once and that they failed to clone ~100 discrete human miRNAs that had been previously documented by others (11). This experience illustrates the rigor and challenges encountered in attempts to validate rare miRNAs which are expressed at low levels.

Human miRNAs are present in introns of coding genes and introns and exons of non-coding transcripts (14). MiRNAs are small ncRNAs of 18 – 25 nucleotides (nt). A mature miRNA emerges after a series of steps that begins with the transcription by RNA polymerase II of a long primary transcript (pri-miRNAs) that contains a “hairpin” structure. Pri-miRNA is first cropped in the nucleus into an ~70 nt stem-loop RNA intermediate (pre-miRNA) by a protein complex that contains the Drosha ribonuclease with an RNA binding protein, known as DGCR8 in humans (15). The pre-miRNA is then ferried by Exportin 5 into the cytoplasm (16), (17) and further processed by a cytoplasmic ribonuclease III enzyme, Dicer, to an imperfectly matched double-stranded mature miRNA (Fig. 1A). One strand of this mature double-stranded miRNA is destined as the guide strand, with the other as the passenger strand. The guide strand is channeled by Dicer-interacting proteins, PACT and TAR RNA-binding

protein (TRBP) (18), (19), (20) into an RNA-induced silencing complex (RISC). Detailed reviews of miRNA biogenesis and RISC complex formation have been presented elsewhere (21), (22).

An miRNA-armed RISC (mi-RISC) represents an effector complex that mediates miRNA function(s) inside cells. In plants, mi-RISC use miRNA-guides which are perfectly complementary to either the coding region or the 3’ untranslated region of cognate mRNAs. In the setting of an miRNA-mRNA interaction driven by perfect complementarity, plant mi-RISC can mediate mRNA cleavage/degradation similar to that described for siRNA (or si-RISC) -mediated silencing (23), (24). By contrast, animal and human mi-RISC recognize target mRNAs using base-pairing in a manner tolerant of mismatches. Thus, in animal cells, the imperfect miRNA - mRNA complementarity is commonly composed of matched nucleotides at positions 2 to 7 (termed the seed sequence) in the 5’ portion of the miRNA (25), (26) (Fig. 1B) with mismatched nucleotides at positions 10 and 11. These mismatches are thought to preclude the endonucleolytic cleavage of mRNA, a phenomenon normally observed with si-RISC mediated RNA interference (RNAi), by mi-RISC.

Once a mi-RISC-mRNA interaction forms in a human cell, how does the resulting complex trigger mRNA silencing? As yet, the answer to this question remains incompletely elucidated and somewhat controversial. Nonetheless, current data are compatible with multiple miRNA mechanisms which either repress mRNA translation or enforce premature mRNA decay. There is evidence that mi-RISC/mRNA

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interaction can promote inhibition of translational initiation, increase co-translational degradation of nascent proteins, reduce the elongation rate of translation, and/or increase the rate of mRNA deadenylation [(27), (28), (29), (30), (31), (32) ; reviewed in (33), (34)]. More recent data suggest that the eIF6 component of miRNA-RISC can prevent productive assembly of the 80S ribosome complex (35). Which mechanism operates under what physiological conditions remains contested. However, the subcellular retention of mi-RISC/mRNA in ribosome-free translationally silent cytoplasmic organelles termed Processing bodies (P-bodies) does appear to account for some aspects of silencing (36), (37), (38). Roles played by miRNAs in development and diseases What biological roles are served by the several hundred characterized human miRNAs? Currently, only a handful of human mRNAs have been conclusively validated as specific targets for miRNAs. Extant bioinformatic predictions suggest that a single miRNA through imperfect complementarity can potentially target ~100 different mRNAs (39). A reasonable extrapolation from these predictions argues that up to 30% of all mammalian genes are under some degree of miRNA-regulation (40), (41). Experimental observations do support important physiological roles contributed by miRNAs. For instance, in C. elegans, zebra fish, Drosophila, mice, and humans, miRNA expression occurs with tissue restricted profiles (42), (43), (44), (45), (46), (47) and differential timing during development (48), (49), (50), (51). Both patterns suggest that miRNAs contribute to

morphological development and organogenesis [reviewed in (43), (52)]. Studies that have deliberately depleted Dicer, the RNAse III enzyme critical to miRNA maturation, from zebra fish and mice have been functionally informative. Knock-out of Dicer in zebra fish arrest embryo development 8 days after fertilization (53), and dicer deficient mice lose viability prior to axis formation during gastrulation (54). Conditional depletion of Dicer in mice has shown that general loss of miRNA function(s) affects T-cell development (55), (56), limb formation (57), and organ maturation (58), (59). Some of these results should be interpreted cautiously since Dicer is known to play roles in heterochromatin formation and chromosome segregation in yeast Schizosaccharomyces pombe which apparently does not encode any known miRNAs. Hence, Dicer has functions beyond miRNA processing, and some of the multicellular loss-of-Dicer phenotypes could occur independently of miRNA-effects. The notion that miRNAs are involved in human diseases arises from two sets of observations. A first clue that dysfunction of miRNA-pathway(s) contributes to pathology came from the recognition that humans with mutations in DGCR8 (a Drosha cofactor) or fragile X (a RISC cofactor) suffer respectively from DiGeorge syndrome (60) and mental retardation (61), (62). Second, >50% of human miRNA genes are present at genetic loci (such as fragile sites, common break point regions etc…) implicated in cancers [reviewed in (63)]. Accordingly, miRNA expression patterns are invariably found to be very different in tumor tissues when compared to matched normals. Indeed, in studies of 334 leukemia and 540

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primary tumors, Lu et al. (64) and Volinia et al. (65) respectively observed miRNA cancer signatures that distinguished tumors based on their tissue origin. Other miRNA profiling studies have also substantiated malignancy-specific expression patterns in lung (66), colon (67), breast (68), and heptacellular (69) cancers. A very recent study has added indirect support that miRNA changes are causal, rather than consequential, of cellular transformation (70). How then do miRNA-changes promote human carcinogenesis? The full answer is unknown, but there are several ways that one could consider mechanisms. One perspective posits that some miRNAs are tumor suppressors while other miRNAs are oncogenes (see Table 1); reduced-expression of the former or gained-expression of the latter would confer growth advantage to cells. Experimental data support that miR-15a and miR-16-1 provide tumor suppressor function by targeting Bcl 2 (71), and miR-155 is oncogenic through incompletely understood effects on perhaps PU.1 and C/EBPβ (72). However, one must entertain the possibility of tissue specific determinants of miRNA function when interpreting findings. Thus, intriguingly, although miR-15a and miR-16-1 are down regulated in chronic lymphocytic leukemia (consistent with their postulated tumor suppressor function; (73)), the same miRNAs are paradoxically over-expressed in endocrine pancreatic tumors (74). MiRNA-changes in cancers offer suggestive correlations, which are usually insufficient to prove conclusively their causality for carcinogenesis. Direct validation of causality can, however, emerge from

studying transforming viruses that do not encode viral oncogenes but do integrate near genome loci whose expression drives tumorigenesis. Using such an approach, the first proof of an oncogenic miRNA causal of cancer was miR-155/BIC [reviewed in (75)] which was activated in chicken tumors by retroviral insertion (i.e. avian leukosis virus integration). Over-expression of human miR-155/BIC has subsequently been linked to the development of Hodgkin’s (72), (76) and Burkitt’s (77), (78) lymphoma. Additional evidence for oncogenic miRNAs used by viruses to transform cells comes from examples of integration induced activation of miR-17-92 and miR-106a-363 in SL3-3 murine leukemia virus tumors (79), (80). Separately, findings of increased human papilloma virus insertion at miRNA-containing fragile sites in cervical carcinomas (81) lend added credence to the general contribution of miRNA perturbations to cancers. ncRNAs and mammalian defense against pathogenic infections Separate from cancers, human infectious diseases in the 21st century represent the second leading cause of death, and the leading global burden on disability-adjusted life-years (82). Infections from HIV-AIDS and hepatitis B and C viruses alone cause >3 million deaths annually (83). Accordingly, an understanding of how mammals defend against viral infections (84) and whether such defenses employ ncRNAs is important.

NcRNAs (e.g. RNAi activity) have, in fact, been proposed to confer wide-ranging anti-viral functions in bacteria (85), (86), plants [reviewed in (87)], and animals [reviewed in (88), (89), (90), (91)]. However, some investigators have argued that sequence-

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specific innate immunity in mammals has been replaced by sequence-non-specific dsRNA-triggered interferon (IFN)-based defense (92). While this hypothesis does frame one possible evolutionary scenario, several findings are incongruent with the complete extinction of mammalian sequence-specific RNAi in favor of an IFN-defense. First, while RNAi is effective for silencing the replication of all classes of mammalian viruses [reviewed in (93)], interferon in practical applications has been shown to be modestly efficacious only in treatments of two viral infections [HBV and HCV; (94), (95)]. Second, there is abundant evidence that cellular miRNAs are employed in sequence-specific fashion by primate cells either to restrict or augment (Fig. 2) the replication of viruses such as primate foamy virus type 1 (PFV-1) (96), HCV (97), or the human immunodeficiency virus type 1 (HIV-1) (98), (99), (100). In response, viruses have evolved RNAi-suppressor proteins or decoy RNAs to counter these cellular restrictions [(101); reviewed in (102)]. Third, several recent studies have found that humans and mice do process cellular siRNAs (103), (104) and miwiRNAs (105), another class of small ncRNAs, for sequence-specific defenses against endogenous retroviruses / retrotransposons. Finally, emerging findings implicate the involvement of human miRNAs in inflammatory responses against pathogenic infections (106), (107) (Table 1), providing a further rationale for the conservation of sequence-specific RNA-function in mammalian host-pathogen interaction.

Mammalian viruses appear to encode viral miRNAs (vmiRNAs) (108;109) which are processed functionally in primate cells. This

observation is consistent with the concept that RNAi pathways are generally important and are preserved from bacteria to plants to mammals. However, it has been argued that not all viruses necessarily encode miRNAs and that vmiRNAs may selectively exist only in viruses (e.g. Herpes) with large DNA genomes (92). This notion arose in part because some investigators (108) have failed to predict and clone small viral ncRNAs described by others (110) (111). Nonetheless, we are early in our understanding of vmiRNAs, and this type of failing should be interpreted with circumspection. For instance, three separate studies on vmiRNAs encoded by Herpes simplex virus type 1 (HSV-1) illustrate the potential for discrepant results. Two of the three studies (112), (113) cloned discrete miRNAs that were not predicted by the third study (108) and failed to clone any of the 8 predicted HSV-1 miRNAs (108). Drawing from the experience of identifying and cloning rare cellular miRNAs (11), it seems reasonable that temporal, spatial, and different tissue culture conditions can all influence individual experimental successes at capturing vmiRNAs. Moreover, a recent bioinformatic analysis found that secondary structures frequent in ncRNAs are evolutionarily selected against in coding regions of genomes (114). This suggests that smaller DNA and RNA viruses based simply on size constraints that require greater portions of their genomes for coding purposes are less likely to retain ncRNAs than their larger viral counterparts .

We note that initial nucleotide sequence-specific selection by the cell against highly mutable viruses such as HIV-1 and HCV could quickly reshape viral genome sequences. Viruses may

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mutate to escape restriction and even evolve propitious adaptations [e.g. the emergence of T20 (an anti-viral peptide) dependent HIV-1 replication after initial negative selection against HIV-1 by T20; (115)] to turn negative effects into positive factors (Fig. 2). This type of virus-host, cat-and-mouse, interplay may ultimately limit the antiviral effectiveness of RNAi (116) and could explain how, unlike other viruses, HCV naturally co-opts a cellular miRNA for enhancing, rather than inhibiting, viral replication (97) (Fig. 2).

Perspective The past few years have been an exciting and challenging period for studying ncRNAs, including cellular and viral miRNAs. Although much has been learned, it is a safe prediction that many new rules, principles, and functions of

small RNAs await elucidation. On the horizon are nascent findings that small RNAs may also serve gene activating rather than gene silencing functions (117). Capturing and consolidating new discoveries in the coming days will help advance our knowledge of miRNAs and their potential applications toward addressing human diseases. Acknowledgements We thank Philip Yeung and Ariel Pearl-Jacobvitz for assistance in preparation of figures and text, and Andrew Dayton for a critical reading of manuscript. Due to space limitation, we apologize for the inability to cite many relevant works published by colleagues. Research in KTJ’s laboratory is supported by intramural NIAID funding.

Table 1. Selected examples of human miRNAs and their proposed functional targets

Tumor Suppressors Targets References let 7 Ras, Hmga2 (118)

miR-15a Bcl 2 (71) miR-16-1 Bcl 2 (71) miR-127 Bcl 6 (119)

Tumor Inducers miR-155 PU.1, C/EBPβ (72)

miR-17-92 E2F1 (120) miR-106a R61 (65) miR-21 Tpmi (121)

Inflammation miR-146 IRAK1,

TRAF6 (122)

miR-155 ? (106) let 7a NF 2 (107)

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Figure legends. Fig. 1. miRNA biogenesis and function. A) Processing of primary miRNA transcript (pri-miRNA) by Drosha and pre-miRNA by Dicer is shown. One strand of the miRNA becomes a guide strand and is incorporated into RISC while the other (passenger) strand is discarded. B) Perfect complementarity of miRNA with mRNA specifies cleavage while imperfect hybrid formation leads to translational silencing. Further details regarding proposed mechanisms used by mi-RISC for silencing are discussed in the text. Fig. 2. Several potential interactions between virus and cell. An infecting virus can trigger changes in cellular miRNA (cmiRNA) (123), produce viral miRNA (vmiRNA) or affect miRNA abundance via synthesis of viral proteins. Each effect can lead to positive (+) or negative (-) outcomes on viral replication. Negative effects exerted by cmiRNA on viruses could cause viral genomes to evolve, adapt, and co-opt such interactions in a positive fashion.

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Ben Berkhout and Kuan-Teh JeangRISCy business: microRNAs, pathogenesis, and viruses

published online July 12, 2007J. Biol. Chem. 

  10.1074/jbc.R700023200Access the most updated version of this article at doi:

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