U-rich RNA Research Articles

Human CSTF2 RNA Recognition Motif Domain Binds to a U-Rich RNA Sequence through a Multistep Binding Process.

The RNA recognition motif (RRM) is a conserved and ubiquitous RNA-binding domain that plays essential roles in mRNA splicing, polyadenylation, transport, and stability. RRM domains exhibit remarkable diversity in binding partners, interacting with various sequences of single- and double-stranded RNA, despite their small size and compact fold. During pre-mRNA cleavage and polyadenylation, the RRM domain from CSTF2 recognizes U- or G/U-rich RNA sequences downstream from the cleavage and polyadenylation site to regulate the process. Given the importance of alternative cleavage and polyadenylation in increasing the diversity of mRNAs, the exact mechanism of binding of RNA to the RRM of CSTF2 remains unclear, particularly in the absence of a structure of this RRM bound to a native RNA substrate. Here, we performed a series of NMR titration and spin relaxation experiments, which were complemented by paramagnetic relaxation enhancement measurements and rigid-body docking, to characterize the interactions of the CSTF2 RRM with a U-rich ligand. Our results reveal a multistep binding process involving differences in ps-ns time scale dynamics and potential structural changes, particularly in the C-terminalα-helix. These results provide insights into how the CSTF2 RRM domain binds to U-rich RNA ligands and offer a greater understanding for the molecular basis of the regulation of pre-mRNA cleavage and polyadenylation.

Structural insights into RNA-mediated transcription regulation in bacteria

RNA can regulate its own synthesis without auxiliary proteins. For example, U-rich RNA sequences signal RNA polymerase (RNAP) to pause transcription and are required for transcript release at intrinsic terminators in all kingdoms of life. In contrast, the regulatory RNA putL suppresses pausing and termination in cis. However, how nascent RNA modulates its own synthesis remains largely unknown. We present cryo-EM reconstructions of RNAP captured during transcription of putL variants or an unrelated sequence at a U-rich pause site. Our results suggest how putL suppresses pausing and promotes its synthesis. We demonstrate that transcribing a U-rich sequence, a ubiquitous trigger of intrinsic termination, promotes widening of the RNAP nucleic-acid-binding channel. Widening destabilizes RNAP interactions with DNA and RNA to facilitate transcript dissociation reminiscent of intrinsic transcription termination. Surprisingly, RNAP remains bound to DNA after transcript release. Our results provide the structural framework to understand RNA-mediated intrinsic transcription termination.

Electrostatic Interactions between CSTF2 and pre-mRNA Drive Cleavage and Polyadenylation

Nascent pre-mRNA 3′-end cleavage and polyadenylation (C/P) involves numerous proteins that recognize multiple RNA elements. Human CSTF2 binds to a downstream U- or G/U-rich sequence through its RNA recognition motif (RRM) regulating C/P. We previously reported the only known disease-related CSTF2 RRM mutant (CSTF2D50A) and showed that it changed the on-rate of RNA binding, leading to alternative polyadenylation in brains of mice carrying the same mutation. In this study, we further investigated the role of electrostatic interactions in the thermodynamics and kinetics of RNA binding for the CSTF2 RRM and the downstream consequences for regulation of C/P. By combining mutagenesis with NMR spectroscopy and biophysical assays, we confirmed that electrostatic attraction is the dominant factor in RRM binding to a naturally occurring U-rich RNA sequence. Moreover, we demonstrate that RNA binding is accompanied by an enthalpy-entropy compensation mechanism that is supported by changes in pico-to-nanosecond timescale RRM protein dynamics. We suggest that the dynamic binding of the RRM to U-rich RNA supports the diversity of sequences it encounters in the nucleus. Lastly, in vivo C/P assays demonstrate a competition between fast, high affinity RNA binding and efficient, correct C/P. These results highlight the importance of the surface charge of the RRM in RNA binding and the balance between nascent mRNA binding and C/P in vivo.

Enhanced Triplex Hybridization of DNA and RNA via Syndiotactic Side Chain Presentation in Minimal bPNAs.

General design principles for recognition at noncanonical interfaces of DNA and RNA remain elusive. Triplex hybridization of bifacial peptide nucleic acids (bPNAs) with oligo-T/U DNAs and RNAs is a robust recognition platform that can be used to define structure-function relationships in synthetic triplex formation. To this end, a set of minimal (mw < 1 kD) bPNA variants was synthesized to probe the impact of amino acid secondary structural propensity, stereochemistry, and backbone cyclization on hybridization with short, unstructured T-rich DNA and U-rich RNAs. Thermodynamic parameters extracted from optical melting analyses of bPNA variant hybrids indicated that there are two bPNA backbone modifications that significantly improve hybridization: alternating (d, l) configuration in open-chain dipeptides and homochiral dipeptide cyclization to diketopiperazine. Further, binding to DNA is preferred over RNA for all bPNA variants. Thymine-uracil substitutions in DNA substrates revealed that the methyl group of thymine accounts for 71% of ΔΔGDNA-RNA for open-chain bPNAs but only 40% of ΔΔGDNA-RNA for diketopiperazine bPNA, suggesting a greater sensitivity to RNA conformation and more optimized stacking in the cyclic bPNA. Together, these data reveal pressure points for tuning triplex hybridization at the chiral centers of bPNA, backbone conformation, stacking effects at the base triple, and the nucleic acid substrate itself. A structural blueprint for enhancing bPNA targeting of both DNA and RNA substrates includes syndiotactic base presentation (as found in homochiral diketopiperazines and d, l peptides), expansion of base stacking, and further investigation of bPNA backbone preorganization.

The KH domain facilitates the substrate specificity and unwinding processivity of DDX43 helicase

The K-homology (KH) domain is a nucleic acid–binding domain present in many proteins. Recently, we found that the DEAD-box helicase DDX43 contains a KH domain in its N-terminus; however, its function remains unknown. Here, we purified recombinant DDX43 KH domain protein and found that it prefers binding ssDNA and ssRNA. Electrophoretic mobility shift assay and NMR revealed that the KH domain favors pyrimidines over purines. Mutational analysis showed that the GXXG loop in the KH domain is involved in pyrimidine binding. Moreover, we found that an alanine residue adjacent to the GXXG loop is critical for binding. Systematic evolution of ligands by exponential enrichment, chromatin immunoprecipitation–seq, and cross-linking immunoprecipitation–seq showed that the KH domain binds C-/T-rich DNA and U-rich RNA. Bioinformatics analysis suggested that the KH domain prefers to bind promoters. Using 15N-heteronuclear single quantum coherence NMR, the optimal binding sequence was identified as TTGT. Finally, we found that the full-length DDX43 helicase prefers DNA or RNA substrates with TTGT or UUGU single-stranded tails and that the KH domain is critically important for sequence specificity and unwinding processivity. Collectively, our results demonstrated that the KH domain facilitates the substrate specificity and processivity of the DDX43 helicase.

Solution structure and RNA-binding of a minimal ProQ-homolog from Legionella pneumophila (Lpp1663).

Small regulatory RNAs (sRNAs) play an important role for posttranscriptional gene regulation in bacteria. sRNAs recognize their target messenger RNAs (mRNAs) by base-pairing, which is often facilitated by interactions with the bacterial RNA-binding proteins Hfq or ProQ. The FinO/ProQ RNA-binding protein domain was first discovered in the bacterial repressor of conjugation, FinO. Since then, the functional role of FinO/ProQ-like proteins in posttranscriptional gene regulation was extensively studied in particular in the enterobacteria E. coli and Salmonella enterica and a wide range of sRNA-targets was identified for these proteins. In addition, enterobacterial ProQ homologs also recognize and protect the 3′-ends of a number of mRNAs from exonucleolytic degradation. However, the RNA-binding properties of FinO/ProQ proteins with regard to the recognition of different RNA targets are not yet fully understood. Here, we present the solution NMR structure of the so far functionally uncharacterized ProQ homolog Lpp1663 from Legionella pneumophila as a newly confirmed member and a minimal model system of the FinO/ProQ protein family. In addition, we characterize the RNA-binding preferences of Lpp1663 with high resolution NMR spectroscopy and isothermal titration calorimetry (ITC). Our results suggest a binding preference for single-stranded uridine-rich RNAs in the vicinity of stable stem–loop structures. According to chemical shift perturbation experiments, the single-stranded U-rich RNAs interact mainly with a conserved RNA-binding surface on the concave site of Lpp1663.

The vault RNA of Trypanosoma brucei plays a role in the production of trans-spliced mRNA

The vault ribonucleoprotein (RNP), comprising vault RNA (vtRNA) and telomerase-associated protein 1 (TEP1), is found in many eukaryotes. However, previous studies of vtRNAs, for example in mammalian cells, have failed to reach a definitive conclusion about their function. vtRNAs are related to Y RNAs, which are complexed with Ro protein and influence Ro's function in noncoding RNA (ncRNA) quality control and processing. In Trypanosoma brucei, the small noncoding TBsRNA-10 was first described in a survey of the ncRNA repertoire in this organism. Here, we report that TBsRNA-10 in T. brucei is a vtRNA, based on its association with TEP1 and sequence similarity to those of other known and predicted vtRNAs. We observed that like vtRNAs in other species, TBsRNA-10 is transcribed by RNA polymerase III, which in trypanosomes also generates the spliceosomal U-rich small nuclear RNAs. In T. brucei, spliced leader (SL)-mediated trans-splicing of pre-mRNAs is an obligatory step in gene expression, and we found here that T. brucei's vtRNA is highly enriched in a non-nucleolar locus in the cell nucleus implicated in SL RNP biogenesis. Using a newly developed permeabilized cell system for the bloodstream form of T. brucei, we show that down-regulated vtRNA levels impair trans-spliced mRNA production, consistent with a role of vtRNA in trypanosome mRNA metabolism. Our results suggest a common theme for the functions of vtRNAs and Y RNAs. We conclude that by complexing with their protein-binding partners TEP1 and Ro, respectively, these two RNA species modulate the metabolism of various RNA classes.

Conserved RNA binding activity of a Yin-Yang 1 homologue in the ova of the purple sea urchin Strongylocentrotus purpuratus

Yin-Yang 1 (YY1) is a highly conserved transcription factor possessing RNA-binding activity. A putative YY1 homologue was previously identified in the developmental model organism Strongylocentrotus purpuratus (the purple sea urchin) by genomic sequencing. We identified a high degree of sequence similarity with YY1 homologues of vertebrate origin which shared 100% protein sequence identity over the DNA- and RNA-binding zinc-finger region with high similarity in the N-terminal transcriptional activation domain. SpYY1 demonstrated identical DNA- and RNA-binding characteristics between Xenopus laevis and S. purpuratus indicating that it maintains similar functional and biochemical properties across widely divergent deuterostome species. SpYY1 binds to the consensus YY1 DNA element, and also to U-rich RNA sequences. Although we detected SpYY1 RNA-binding activity in ova lysates and observed cytoplasmic localization, SpYY1 was not associated with maternal mRNA in ova. SpYY1 expressed in Xenopus oocytes was excluded from the nucleus and associated with maternally expressed cytoplasmic mRNA molecules. These data demonstrate the existence of an YY1 homologue in S. purpuratus with similar structural and biochemical features to those of the well-studied vertebrate YY1; however, the data reveal major differences in the biological role of YY1 in the regulation of maternally expressed mRNA in the two species.

Long Non-Coding RNAs: Novel Players in Regulation of Immune Response Upon Herpesvirus Infection.

Herpesviruses have developed a variety of sophisticated immune evasion strategies to establish lifelong latent infection, including the use of long non-coding RNAs (lncRNAs). In this review, we summarize the lncRNA action modes, i.e., RNA–protein, RNA–RNA, and RNA–DNA interactions, involved in regulating important aspects of immunity by controlling gene expression at various stages. Upon herpesvirus infection, host lncRNAs, such as nuclear paraspeckle assembly transcript 1, negative regulator of antiviral, and B-cell integration cluster have been functionally characterized as negative or positive antiviral regulators in the immune response. Herpesviruses have also evolved multiple strategies to modulate the host immune response using lncRNAs, such as latency-associated transcript, β 2.7 RNA, 5 kb and 7.2 kb lncRNAs, Epstein–Barr virus-encoded non-coding RNA, BamH I-A rightward transcripts, polyadenylated nuclear, and herpesvirus saimiri U-rich RNAs. We discuss the various mechanisms of immune-related lncRNAs, and their diversified and important functions in the modulation of innate and adaptive immunity upon herpesvirus infection as well as in host–pathogen interactions, which will facilitate our understanding of rational design of novel strategies to combat herpesvirus infection.

Hsp70's RNA-binding and mRNA-stabilizing activities are independent of its protein chaperone functions

Hsp70 is a protein chaperone that prevents protein aggregation and aids protein folding by binding to hydrophobic peptide domains through a reversible mechanism directed by an ATPase cycle. However, Hsp70 also binds U-rich RNA including some AU-rich elements (AREs) that regulate the decay kinetics of select mRNAs and has recently been shown to bind and stabilize some ARE-containing transcripts in cells. Previous studies indicated that both the ATP- and peptide-binding domains of Hsp70 contributed to the stability of Hsp70-RNA complexes and that ATP might inhibit RNA recruitment. This suggested the possibility that RNA binding by Hsp70 might mimic features of its peptide-directed chaperone activities. Here, using purified, cofactor-free preparations of recombinant human Hsp70 and quantitative biochemical approaches, we found that high-affinity RNA binding requires at least 30 nucleotides of RNA sequence but is independent of Hsp70's nucleotide-bound status, ATPase activity, or peptide-binding roles. Furthermore, although both the ATP- and peptide-binding domains of Hsp70 could form complexes with an ARE sequence from VEGFA mRNA in vitro, only the peptide-binding domain could recover cellular VEGFA mRNA in ribonucleoprotein immunoprecipitations. Finally, Hsp70-directed stabilization of VEGFA mRNA in cells was mediated exclusively by the protein's peptide-binding domain. Together, these findings indicate that the RNA-binding and mRNA-stabilizing functions of Hsp70 are independent of its protein chaperone cycle but also provide potential mechanical explanations for several well-established and recently discovered cytoprotective and RNA-based Hsp70 functions.

Will the circle be unbroken: specific mutations in the yeast Sm protein ring expose a requirement for assembly factor Brr1, a homolog of Gemin2.

A seven-subunit Sm protein ring assembles around specific U-rich RNA segments of the U1, U2, U4, and U5 snRNPs that direct pre-mRNA splicing. Using human snRNP crystal structures to guide mutagenesis in Saccharomyces cerevisiae, we gained new insights into structure–function relationships of the SmD1 and SmD2 subunits. Of 18 conserved amino acids comprising their RNA-binding sites or intersubunit interfaces, only Arg88 in SmD1 and Arg97 in SmD2 were essential for growth. Tests for genetic interactions with non-Sm splicing factors identified benign mutations of SmD1 (N37A, R88K, R93A) and SmD2 (R49A, N66A, R97K, D99A) that were synthetically lethal with null alleles of U2 snRNP subunits Lea1 and/or Msl1. Tests of 264 pairwise combinations of SmD1 and SmD2 alleles with each other and with a collection of SmG, SmE, SmF, SmB, and SmD3 alleles revealed 92 instances of inter-Sm synthetic lethality. We leveraged the Sm mutant collection to illuminate the function of the yeast Sm assembly factor Brr1 and its relationship to the metazoan Sm assembly factor Gemin2. Mutations in the adjacent SmE (K83A), SmF (K32A, F33A, R74K), SmD2 (R49A, N66A, E74A, R97K, D99A), and SmD1 (E18A, N37A) subunits—but none in the SmG, SmD3, and SmB subunits—were synthetically lethal with brr1Δ. Using complementation of brr1Δ lethality in two Sm mutant backgrounds as an in vivo assay of Brr1 activity, we identified as essential an N-terminal segment of Brr1 (amino acids 24–47) corresponding to the Gemin2 α1 helix that interacts with SmF and a Brr1 C-terminal peptide (336QKDLIE341) that, in Gemin2, interacts with SmD2.

Anticodon-like binding of the HIV-1 tRNA-like element to human lysyl-tRNA synthetase.

A critical step in the HIV-1 lifecycle involves reverse transcription of the viral genomic RNA (gRNA). Human tRNALys3 serves as a primer for transcription initiation and is selectively enriched in virus particles. Human lysyl-tRNA synthetase (hLysRS) is also packaged into virions. Recently, a tRNA-like element (TLE) within the HIV-1 gRNA was shown to mimic the global tRNA fold and bind competitively to hLysRS, suggesting a mechanism of tRNA targeting to the primer binding site (PBS) and release from the synthetase. Here, we use NMR to investigate hLysRS anticodon-binding domain (ACB) binding to six RNA oligonucleotides, including a hairpin derived from the HIV-1 gRNA TLE. We show that ACB interacts with submicromolar affinity to U-rich RNA oligonucleotides—the tRNALys3 anticodon stem–loop (ACSL), the WT TLE, and a nonanucleotide, U9. In contrast, the ACB bound only weakly to two TLE loop mutants and a C9 nonanucleotide. NMR chemical shift perturbations induced by each RNA indicate that the ACSL and the WT TLE both interact with the ACB in a strikingly similar manner. Taken together, these findings support the conclusion that tRNA mimicry by the HIV-1 genome leads to a highly specific protein–RNA interaction that facilitates efficient primer release from hLysRS prior to reverse transcription.

Rice MEL2, the RNA recognition motif (RRM) protein, binds in vitro to meiosis-expressed genes containing U-rich RNA consensus sequences in the 3'-UTR.

Post-transcriptional gene regulation by RNA recognition motif (RRM) proteins through binding to cis-elements in the 3'-untranslated region (3'-UTR) is widely used in eukaryotes to complete various biological processes. Rice MEIOSIS ARRESTED AT LEPTOTENE2 (MEL2) is the RRM protein that functions in the transition to meiosis in proper timing. The MEL2 RRM preferentially associated with the U-rich RNA consensus, UUAGUU[U/A][U/G][A/U/G]U, dependently on sequences and proportionally to MEL2 protein amounts in vitro. The consensus sequences were located in the putative looped structures of the RNA ligand. A genome-wide survey revealed a tendency of MEL2-binding consensus appearing in 3'-UTR of rice genes. Of 249 genes that conserved the consensus in their 3'-UTR, 13 genes spatiotemporally co-expressed with MEL2 in meiotic flowers, and included several genes whose function was supposed in meiosis; such as Replication protein A and OsMADS3. The proteome analysis revealed that the amounts of small ubiquitin-related modifier-like protein and eukaryotic translation initiation factor3-like protein were dramatically altered in mel2 mutant anthers. Taken together with transcriptome and gene ontology results, we propose that the rice MEL2 is involved in the translational regulation of key meiotic genes on 3'-UTRs to achieve the faithful transition of germ cells to meiosis.

Herpesvirus saimiri MicroRNAs Preferentially Target Host Cell Cycle Regulators

In latently infected marmoset T cells, Herpesvirus saimiri (HVS) expresses six microRNAs (known as miR-HSURs [H. saimiri U-rich RNAs]). The viral miR-HSURs are processed from chimeric primary transcripts, each containing a noncoding U-rich RNA (HSUR) and a pre-miRNA hairpin. To uncover the functions of miR-HSURs, we identified mRNA targets in infected cells using high-throughput sequencing of RNA isolated by cross-linking immunoprecipitation (HITS-CLIP). HITS-CLIP revealed hundreds of robust Argonaute (Ago) binding sites mediated by miR-HSURs that map to the host genome but few in the HVS genome. Gene ontology analysis showed that several pathways regulating the cell cycle are enriched among cellular targets of miR-HSURs. Interestingly, miR-HSUR4-3p represses expression of the p300 transcriptional coactivator by binding the open reading frame of its mRNA. miR-HSUR5-3p directly regulates BiP, an endoplasmic reticulum (ER)-localized chaperone facilitating maturation of major histocompatibility complex class I (MHC-I) and the antiviral response. miR-HSUR5-3p also robustly downregulates WEE1, a key negative regulator of cell cycle progression, leading to reduced phosphorylation of its substrate, cyclin-dependent kinase (Cdk1). Consistently, inhibition of miR-HSUR5-3p in HVS-infected cells decreases their proliferation. Together, our results shed light on the roles of viral miRNAs in cellular transformation and viral latency. Viruses express miRNAs during various stages of infection, suggesting that viral miRNAs play critical roles in the viral life cycle. Compared to protein-coding genes, the functions of viral miRNAs are not well understood. This is because it has been challenging to identify their mRNA targets. Here, we focused on the functions of the recently discovered HVS miRNAs, called miR-HSURs. HVS is an oncogenic gammaherpesvirus that causes acute T-cell lymphomas and leukemias in New World primates and transforms human T cells. A better understanding of HVS biology will help advance our knowledge of virus-induced oncogenesis. Because numerous cellular miRNAs play crucial roles in cancer, viral miRNAs from the highly oncogenic HVS might also be important for transformation. Here, we found that the miR-HSURs preferentially modulate expression of host cell cycle regulators, as well as antiviral response factors. Our work provides further insight into the functions of herpesviral miRNAs in virus-induced oncogenesis and latency.

RNPs and autoimmunity: 20 years later.

RNPs and autoimmunity: 20 years later.

The C-terminal RNA binding motif of HuR is a multi-functional domain leading to HuR oligomerization and binding to U-rich RNA targets

Human antigen R (HuR) is a 32 kDa protein with 3 RNA Recognition Motifs (RRMs), which bind to Adenylate and uridylate Rich Elements (AREs) of mRNAs. Whereas the N-terminal and central domains (RRM1 and RRM2) are essential for AREs recognition, little is known on the C-terminal RRM3 beyond its implication in HuR oligomerization and apoptotic signaling. We have developed a detergent-based strategy to produce soluble RRM3 for structural studies. We have found that it adopts the typical RRM fold, does not interact with the RRM1 and RRM2 modules, and forms dimers in solution. Our NMR measurements, combined with Molecular Dynamics simulations and Analytical Ultracentrifugation experiments, show that the protein dimerizes through a helical region that contains the conserved W261 residue. We found that HuR RRM3 binds to 5′-mer U-rich RNA stretches through the solvent exposed side of its β-sheet, located opposite to the dimerization site. Upon mimicking phosphorylation by the S318D replacement, RRM3 mutant shows less ability to recognize RNA due to an electrostatic repulsion effect with the phosphate groups. Our study brings new insights of HuR RRM3 as a domain involved in protein oligomerization and RNA interaction, both functions regulated by 2 surfaces on opposite sides of the RRM domain.

Recognition of U-rich RNA by Hfq from the Gram-positive pathogen Listeria monocytogenes.

Hfq is a post-transcriptional regulator that binds U- and A-rich regions of sRNAs and their target mRNAs to stimulate their annealing in order to effect translation regulation and, often, to alter their stability. The functional importance of Hfq and its RNA-binding properties are relatively well understood in Gram-negative bacteria, whereas less is known about the RNA-binding properties of this riboregulator in Gram-positive species. Here, we describe the structure of Hfq from the Gram-positive pathogen Listeria monocytogenes in its RNA-free form and in complex with a U6 oligoribonucleotide. As expected, the protein takes the canonical hexameric toroidal shape of all other known Hfq structures. The U6 RNA binds on the “proximal face” in a pocket formed by conserved residues Q9, N42, F43, and K58. Additionally residues G5 and Q6 are involved in protein-nucleic and inter-subunit contacts that promote uracil specificity. Unlike Staphylococcus aureus (Sa) Hfq, Lm Hfq requires magnesium to bind U6 with high affinity. In contrast, the longer oligo-uridine, U16, binds Lm Hfq tightly in the presence or absence of magnesium, thereby suggesting the importance of additional residues on the proximal face and possibly the lateral rim in RNA interaction. Intrinsic tryptophan fluorescence quenching (TFQ) studies reveal, surprisingly, that Lm Hfq can bind (GU)3G and U6 on its proximal and distal faces, indicating a less stringent adenine-nucleotide specificity site on the distal face as compared to the Gram-positive Hfq proteins from Sa and Bacillus subtilis and suggesting as yet uncharacterized RNA-binding modes on both faces.

HumanViCe: host ceRNA network in virus infected cells in human.

Host-virus interaction via host cellular components has been an important field of research in recent times. RNA interference mediated by short interfering RNAs and microRNAs (miRNA), is a widespread anti-viral defense strategy. Importantly, viruses also encode their own miRNAs. In recent times miRNAs were identified as key players in host-virus interaction. Furthermore, viruses were shown to exploit the host miRNA networks to suite their own need. The complex cross-talk between host and viral miRNAs and their cellular and viral targets forms the environment for viral pathogenesis. Apart from protein-coding mRNAs, non-coding RNAs may also be targeted by host or viral miRNAs in virus infected cells, and viruses can exploit the host miRNA mediated gene regulatory network via the competing endogenous RNA effect. A recent report showed that viral U-rich non-coding RNAs called HSUR, expressed in primate virus herpesvirus saimiri (HVS) infected T cells, were able to bind to three host miRNAs, causing significant alteration in cellular level for one of the miRNAs. We have predicted protein coding and non protein-coding targets for viral and human miRNAs in virus infected cells. We identified viral miRNA targets within host non-coding RNA loci from AGO interacting regions in three different virus infected cells. Gene ontology (GO) and pathway enrichment analysis of the genes comprising the ceRNA networks in the virus infected cells revealed enrichment of key cellular signaling pathways related to cell fate decisions and gene transcription, like Notch and Wnt signaling pathways, as well as pathways related to viral entry, replication and virulence. We identified a vast number of non-coding transcripts playing as potential ceRNAs to the immune response associated genes; e.g., APOBEC family genes, in some virus infected cells. All these information are compiled in HumanViCe (http://gyanxet-beta.com/humanvice), a comprehensive database that provides the potential ceRNA networks in virus infected human cells.

The binding of TIA-1 to RNA C-rich sequences is driven by its C-terminal RRM domain

T-cell intracellular antigen-1 (TIA-1) is a key DNA/RNA binding protein that regulates translation by sequestering target mRNAs in stress granules (SG) in response to stress conditions. TIA-1 possesses three RNA recognition motifs (RRM) along with a glutamine-rich domain, with the central domains (RRM2 and RRM3) acting as RNA binding platforms. While the RRM2 domain, which displays high affinity for U-rich RNA sequences, is primarily responsible for interaction with RNA, the contribution of RRM3 to bind RNA as well as the target RNA sequences that it binds preferentially are still unknown. Here we combined nuclear magnetic resonance (NMR) and surface plasmon resonance (SPR) techniques to elucidate the sequence specificity of TIA-1 RRM3. With a novel approach using saturation transfer difference NMR (STD-NMR) to quantify protein–nucleic acids interactions, we demonstrate that isolated RRM3 binds to both C- and U-rich stretches with micromolar affinity. In combination with RRM2 and in the context of full-length TIA-1, RRM3 significantly enhanced the binding to RNA, particularly to cytosine-rich RNA oligos, as assessed by biotinylated RNA pull-down analysis. Our findings provide new insight into the role of RRM3 in regulating TIA-1 binding to C-rich stretches, that are abundant at the 5′ TOPs (5′ terminal oligopyrimidine tracts) of mRNAs whose translation is repressed under stress situations.

Structural and Functional Studies of a Novel RNA-Binding Sm-Like Archaeal Protein

In all three domains of life, Sm oligomers interact with RNA, either as chaperones or as scaffolding to facilitate RNA···RNA and RNA···protein interactions. In bacteria and eukarya, Sm homologs are key for RNA metabolism, for instance in RNA-mediated post-transcriptional regulation in bacteria. However, the in vivo roles of Sm-like archaeal proteins (SmAPs) remain unknown. Sequence searches suggest that the hyperthermophilic crenarchaeote Pyrobaculum aerophilum (Pae) encodes three SmAPs. The two known Pae Sm structures illuminated Sm protein evolution and assembly, and implied that these homologs may represent an ancestral form of the heteromeric Sm complexes that pervade eukaryotic RNA processing, e.g. in the spliceosome and other RNPs. We have now characterized Pae SmAP2, the final putative Sm paralog from this species. Biophysical characterization suggests that this SmAP2 exists in multiple stable oligomeric states, which we also detect via chemical cross-linking and mass spectrometry. We have now determined the crystal structure of this SmAP in multiple space-groups, revealing unexpected and hitherto unseen Sm assembly states. Binding assays with various small RNA oligonucleotides show this SmAP to be able to bind U-rich RNA, but not adenine- or cytosine-rich RNAs.