Shaping the host cell environment with viral noncoding RNAs.

Just like the cells they infect viruses express different classes of noncoding RNAs (ncRNAs). Viral ncRNAs come in all shapes and forms, and they usually associate with cellular proteins that are important for their functions. Viral ncRNAs have diverse functions, but they all contribute to the viral control of the cellular environment. Viruses utilize ncRNAs to regulate viral replication, to decide whether they should remain latent or reactivate, to evade the host immune responses, or to promote cellular transformation. In this review we describe the diverse functions played by different classes of ncRNAs expressed by adenoviruses and herpesviruses, how they contribute to the viral infection, and how their study led to insights into RNA-based mechanisms at play in host cells.

Allosteric regulation of noncoding RNA function by microRNAs.

HSUR1 and HSUR2, two noncoding RNAs expressed by the oncogenic Herpesvirus saimiri, bind host microRNAs miR-142-3p, miR-16, and miR-27 with different purposes. While binding of miR-27 to HSUR1 triggers the degradation of the microRNA, miR-16 is tethered by HSUR2 to target host mRNAs to repress their expression. Here we show that the interaction with miR-142-3p is required for the activity of both HSURs. Coimmunoprecipitation experiments revealed that miR-142-3p allosterically regulates the binding of miR-27 and miR-16 to HSUR1 and HSUR2, respectively. The binding of two different miRNAs to each HSUR is not cooperative. HSURs can be engineered to be regulated by other miRNAs, indicating that the identity of the binding miRNA is not important for HSUR regulation. Our results uncover a mechanism for allosteric regulation of noncoding RNA function and a previously unappreciated way in which microRNAs can regulate gene expression.

Learning noncoding RNA biology from viruses.

Insights into interactions between viral factors and the cellular machinery usually lead to discoveries concerning host cell biology. Thus, the gene expression field has historically relied on viral model systems to discover mechanisms underlying different cellular processes. In recent years, the functional characterization of the small nuclear noncoding RNAs expressed by the oncogenic Herpesvirus saimiri, called HSURs, resulted in the discovery of two mechanisms for the regulation of gene expression. HSUR1 and HSUR2 associate with host microRNAs, which are small noncoding RNAs that broadly regulate gene expression by binding to messenger RNAs. HSUR1 provided the first example of a process known as target-directed miRNA degradation that operates in cells to regulate miRNA populations. HSUR2 functions as a miRNA adaptor, uncovering an entirely new, indirect mechanism by which miRNAs can inhibit mRNA function. Here, I review the path that led to these discoveries and their implications and postulate new exciting questions about the functions of these fascinating viral noncoding RNAs.

Viral miRNA adaptor differentially recruits miRNAs to target mRNAs through alternative base-pairing.

HSUR2 is a viral non-coding RNA (ncRNA) that functions as a microRNA (miRNA) adaptor. HSUR2 inhibits apoptosis in infected cells by recruiting host miRNAs miR-142-3p and miR-16 to mRNAs encoding apoptotic factors. HSUR2's target recognition mechanism is not understood. It is also unknown why HSUR2 utilizes miR-16 to downregulate only a subset of transcripts. We developed a general method for individual-nucleotide resolution RNA-RNA interaction identification by crosslinking and capture (iRICC) to identify sequences mediating interactions between HSUR2 and target mRNAs in vivo. Mutational analyses confirmed identified HSUR2-mRNA interactions and validated iRICC as a method that confidently determines sequences mediating RNA-RNA interactions in vivo. We show that HSUR2 does not display a 'seed' region to base-pair with most target mRNAs, but instead uses different regions to interact with different transcripts. We further demonstrate that this versatile mode of interaction via variable base-pairing provides HSUR2 with a mechanism for differential miRNA recruitment.

Differential transcriptional profiles identify microglial- and macrophage-specific gene markers expressed during virus-induced neuroinflammation.

BACKGROUND: In the healthy central nervous system (CNS), microglia are found in a homeostatic state and peripheral macrophages are absent from the brain. Microglia play key roles in maintaining CNS homeostasis and acting as first responders to infection and inflammation, and peripheral macrophages infiltrate the CNS during neuroinflammation. Due to their distinct origins and functions, discrimination between these cell populations is essential to the comprehension of neuroinflammatory disorders. Studies comparing the gene profiles of microglia and peripheral macrophages, or macrophages in vitro-derived from bone marrow, under non-infectious conditions of the CNS, have revealed valuable microglial-specific genes. However, studies comparing gene profiles between CNS-infiltrating macrophages and microglia, when both are isolated from the CNS during viral-induced neuroinflammation, are lacking. METHODS: We isolated, via flow cytometry, microglia and infiltrating macrophages from the brains of Theiler's murine encephalomyelitis virus-infected C57BL/6 J mice and used RNA-Seq, followed by validation with qPCR, to examine the differential transcriptional profiles of these cells. We utilized primary literature defining subcellular localization to determine whether or not particular proteins extracted from the transcriptional profiles were expressed at the cell surface. The surface expression and cellular specificity of triggering receptor expressed on myeloid cells 1 (TREM-1) protein were examined via flow cytometry. We also examined the immune response gene profile within the transcriptional profiles of these isolated microglia and infiltrating macrophages. RESULTS: We have identified and validated new microglial- and macrophage-specific genes, encoding cell surface proteins, expressed at the peak of neuroinflammation. TREM-1 protein was confirmed to be expressed by infiltrating macrophages, not microglia, at the peak of neuroinflammation. We also identified both unique and redundant immune functions, through examination of the immune response gene profiles, of microglia and infiltrating macrophages during neurotropic viral infection. CONCLUSIONS: The differential expression of cell surface-specific genes during neuroinflammation can potentially be used to discriminate between microglia and macrophages as well as provide a resource that can be further utilized to target and manipulate specific cell responses during neuroinflammation.

Novel roles for Sm-class RNAs in the regulation of gene expression.

Viruses masterfully regulate host gene expression during infection. Many do so, in part, by expressing non-coding RNAs. Recent work has shown that HSUR 2, a viral non-coding RNA expressed by the oncogenic Herpesvirus saimiri, regulates mRNA expression through a novel mechanism. HSUR 2 base pairs with both target mRNAs and host miRNAs in infected cells. This results in HSUR 2-dependent recruitment of host miRNAs and associated Ago proteins to target mRNAs, and the subsequent destabilization of target mRNAs. Using this mechanism, this virus regulates key cellular pathways during viral infection. Here I discuss the evolution of our thinking about HSUR function and explore the implications of recent findings in relation to the current views on the functions of interactions between miRNAs and other classes of non-coding RNAs, the potential advantages of this mechanism of regulation of gene expression, and the evolutionary origin of HSUR 2.

A viral Sm-class RNA base-pairs with mRNAs and recruits microRNAs to inhibit apoptosis.

Viruses express several classes of non-coding RNAs; the functions and mechanisms by which most of these act are unknown. Herpesvirus saimiri, a γ-herpesvirus that establishes latency in the T cells of New World primates and has the ability to cause aggressive leukaemias and lymphomas in non-natural hosts, expresses seven small nuclear uracil-rich non-coding RNAs (called HSURs) in latently infected cells. These HSURs associate with Sm proteins, and share biogenesis and structural features with cellular Sm-class small nuclear RNAs. One of these HSURs (HSUR2) base-pairs with two host cellular microRNAs (miR-142-3p and miR-16) but does not affect their abundance or activity, which suggests that its interactions with them perform alternative functions. Here we show that HSUR2 also base-pairs with mRNAs in infected cells. We combined in vivo psoralen-mediated RNA-RNA crosslinking and high-throughput sequencing to identify the mRNAs targeted by HSUR2, which include mRNAs that encode retinoblastoma and factors involved in p53 signalling and apoptosis. We show that HSUR2 represses the expression of target mRNAs and that base-pairing between HSUR2 and miR-142-3p and miR-16 is essential for this repression, suggesting that HSUR2 recruits these two cellular microRNAs to its target mRNAs. Furthermore, we show that HSUR2 uses this mechanism to inhibit apoptosis. Our results uncover a role for this viral Sm-class RNA as a microRNA adaptor in the regulation of gene expression that follows precursor mRNA processing.

MicroRNA-9 controls dendritic development by targeting REST.

MicroRNAs (miRNAs) are conserved noncoding RNAs that function as posttranscriptional regulators of gene expression. miR-9 is one of the most abundant miRNAs in the brain. Although the function of miR-9 has been well characterized in neural progenitors, its role in dendritic and synaptic development remains largely unknown. In order to target miR-9 in vivo, we developed a transgenic miRNA sponge mouse line allowing conditional inactivation of the miR-9 family in a spatio-temporal-controlled manner. Using this novel approach, we found that miR-9 controls dendritic growth and synaptic transmission in vivo. Furthermore, we demonstrate that miR-9-mediated downregulation of the transcriptional repressor REST is essential for proper dendritic growth.

RNase III-independent microRNA biogenesis in mammalian cells.

RNase III enzymes are fundamental to the biogenesis of microRNAs (miRNAs) and small interfering RNAs (siRNAs) in all species studied. Although alternative miRNA pathways independent of Drosha or Dicer exist, each still requires one RNase III-type enzyme. Here, we describe two strategies that marry either RNase Z or the Integrator complex with the slicing activity of Argonaute2 to generate highly functional mature miRNAs. We provide stringent validation of their RNase III independence by demonstrating efficient miRNA biogenesis and activity in Drosha and Dicer knockout cells. These data provide proof-of-principle evidence for additional mechanistic possibilities for efficient generation of small regulatory RNAs, and represent novel silencing triggers that may be exploited for technical purposes.

A primate herpesvirus uses the integrator complex to generate viral microRNAs.

Herpesvirus saimiri (HVS) is a γ-herpesvirus that expresses Sm class U RNAs (HSURs) in latently infected marmoset T cells. By deep sequencing, we identified six HVS microRNAs (miRNAs) that are derived from three hairpin structures located immediately downstream of the 3' end processing signals of three of the HSURs. The viral miRNAs associate with Ago proteins and are biologically active. We confirmed that the expression of the two classes of viral noncoding RNAs is linked by identifying chimeric HSUR-pre-miRNA transcripts. We show that HVS miRNA biogenesis relies on cis-acting elements specifically required for synthesis and processing of Sm class RNAs. Knockdown of protein components in vivo and processing assays in vitro demonstrated that HVS does not utilize the Microprocessor complex that generates most host miRNAs. Instead, the Integrator complex cleaves to generate the 3' end of the HSUR and the pre-miRNA hairpin. Exportin-5 and Dicer are then required to generate mature viral miRNAs.

Noncoding RNPs of viral origin.

Like their host cells, many viruses produce noncoding (nc)RNAs. These show diversity with respect to time of expression during viral infection, length and structure, protein-binding partners and relative abundance compared with their host-cell counterparts. Viruses, with their limited genomic capacity, presumably evolve or acquire ncRNAs only if they selectively enhance the viral life cycle or assist the virus in combating the host's response to infection. Despite much effort, identifying the functions of viral ncRNAs has been extremely challenging. Recent technical advances and enhanced understanding of host-cell ncRNAs promise accelerated insights into the RNA warfare mounted by this fascinating class of RNPs.

miR-29 and miR-30 regulate B-Myb expression during cellular senescence.

Cellular senescence is a form of irreversible growth arrest and a major tumor suppressor mechanism. We show here that the miR-29 and miR-30 microRNA families are up-regulated during induced and replicative senescence and that up-regulation requires activation of the Rb pathway. Expression of a reporter construct containing the 3'UTR of the B-Myb oncogene is repressed during senescence, and repression is blocked by mutations in conserved miR-29 and miR-30 binding sites in the B-Myb 3'UTR. In proliferating cells, transfection of miR-29 and miR-30 represses a reporter construct containing the wild-type but not the mutant B-Myb 3'UTR, and repression of the mutant 3'UTR is reinstituted by compensatory mutations in miR-29 and miR-30 that restore binding to the mutant sites. miR-29 and miR-30 introduction also represses expression of endogenous B-Myb and inhibits cellular DNA synthesis. Finally, interference with miR-29 and miR-30 expression inhibits senescence. These findings demonstrate that miR-29 and miR-30 regulate B-Myb expression by binding to its 3'UTR and suggest that these microRNAs play an important role in Rb-driven cellular senescence.

Down-regulation of a host microRNA by a Herpesvirus saimiri noncoding RNA.

T cells transformed by Herpesvirus saimiri express seven viral U-rich noncoding RNAs of unknown function called HSURs. We noted that conserved sequences in HSURs 1 and 2 constitute potential binding sites for three host-cell microRNAs (miRNAs). Coimmunoprecipitation experiments confirmed that HSURs 1 and 2 interact with the predicted miRNAs in virally transformed T cells. The abundance of one of these miRNAs, miR-27, is dramatically lowered in transformed cells, with consequent effects on the expression of miR-27 target genes. Transient knockdown and ectopic expression of HSUR 1 demonstrate that it directs degradation of mature miR-27 in a sequence-specific and binding-dependent manner. This viral strategy illustrates use of a ncRNA to manipulate host-cell gene expression via the miRNA pathway.

Reversible phosphorylation differentially affects nuclear and cytoplasmic functions of splicing factor 2/alternative splicing factor.

The Ser/Arg-rich (SR) proteins constitute a family of highly conserved nuclear phosphoproteins that are involved in many steps of mRNA metabolism. Previously, we demonstrated that shuttling SR proteins can associate with translating ribosomes and enhance translation of reporter mRNAs both in vivo and in vitro. Here, we show that endogenous, cytoplasmic splicing factor 2/alternative splicing factor (SF2/ASF) associated with the translation machinery is hypophosphorylated, suggesting that the phosphorylation state of the Arg-Ser-rich (RS) domain may influence the role of SF2/ASF in cytoplasmic RNA processing. In agreement, we show that mutations mimicking a hypophosphorylated RS domain strongly increased SF2/ASF binding to cytoplasmic mRNA and its activity in translation. We also demonstrate that, whereas the RS domain is not required for the function of SF2/ASF in mRNA translation in vivo or in vitro, its second RNA recognition motif (RRM)2 plays a critical role in this process. Taken together, these data suggest that RS-domain phosphorylation may influence the association of SF2/ASF with mRNA, whereas RRM2 may play an important role in mediating protein-protein interactions during translation. These data are consistent with a model whereby reversible protein phosphorylation differentially regulates the subcellular localization and activity of shuttling SR proteins.

A rapid and efficient protocol to purify biologically active recombinant proteins from mammalian cells.

Here, we describe a simple and efficient method for the expression and purification of active recombinant proteins in mammalian cells. This method uses the expression of T7 epitope-tagged proteins in transiently transfected 293T cells grown in monolayer, followed by anti-T7-agarose affinity chromatography. This procedure yields approximately between 75 and 100 microg of biologically active protein/150 cm(2) flask that can be used for biochemical studies. We have tested this protocol for the expression of the prototype SR protein, SF2/ASF, which is a member of the SR protein family with a role in constitutive and alternative splicing. We show that SF2/ASF purified using this protocol is able to complement an S100 HeLa extract, demonstrating that is biologically active. Moreover, expression of a novel SR-related protein that it is required for the second step of pre-mRNA splicing also rendered an active protein. In summary, we present a protocol based on transient transfection of mammalian cells that results in easy purification of significant amounts of biologically active proteins.

A novel SR-related protein is required for the second step of Pre-mRNA splicing.

The SR family proteins and SR-related polypeptides are important regulators of pre-mRNA splicing. A novel SR-related protein of an apparent molecular mass of 53 kDa was isolated in a gene trap screen that identifies proteins which localize to the nuclear speckles. This novel protein possesses an arginine- and serine-rich domain and was termed SRrp53 (for SR-related protein of 53 kDa). In support for a role of this novel RS-containing protein in pre-mRNA splicing, we identified the mouse ortholog of the Saccharomyces cerevisiae U1 snRNP-specific protein Luc7p and the U2AF65-related factor HCC1 as interacting proteins. In addition, SRrp53 is able to interact with some members of the SR family of proteins and with U2AF35 in a yeast two-hybrid system and in cell extracts. We show that in HeLa nuclear extracts immunodepleted of SRrp53, the second step of pre-mRNA splicing is blocked, and recombinant SRrp53 is able to restore splicing activity. SRrp53 also regulates alternative splicing in a concentration-dependent manner. Taken together, these results suggest that SRrp53 is a novel SR-related protein that has a role both in constitutive and in alternative splicing.

Nuclear export and retention signals in the RS domain of SR proteins.

Splicing factors of the SR protein family share a modular structure consisting of one or two RNA recognition motifs (RRMs) and a C-terminal RS domain rich in arginine and serine residues. The RS domain, which is extensively phosphorylated, promotes protein-protein interactions and directs subcellular localization and-in certain situations-nucleocytoplasmic shuttling of individual SR proteins. We analyzed mutant versions of human SF2/ASF in which the natural RS repeats were replaced by RD or RE repeats and compared the splicing and subcellular localization properties of these proteins to those of SF2/ASF lacking the entire RS domain or possessing a minimal RS domain consisting of 10 consecutive RS dipeptides (RS10). In vitro splicing of a pre-mRNA that requires an RS domain could take place when the mutant RD, RE, or RS10 domain replaced the natural domain. The RS10 version of SF2/ASF shuttled between the nucleus and the cytoplasm in the same manner as the wild-type protein, suggesting that a tract of consecutive RS dipeptides, in conjunction with the RRMs of SF2/ASF, is necessary and sufficient to direct nucleocytoplasmic shuttling. However, the SR protein SC35 has two long stretches of RS repeats, yet it is not a shuttling protein. We demonstrate the presence of a dominant nuclear retention signal in the RS domain of SC35.