STAU2 binds a complex RNA cargo that changes temporally with production of diverse intermediate progenitor cells during mouse corticogenesis.

STAU2 is a double-stranded RNA-binding protein enriched in the nervous system. During asymmetric divisions in the developing mouse cortex, STAU2 preferentially distributes into the intermediate progenitor cell (IPC), delivering RNA molecules that can impact IPC behavior. Corticogenesis occurs on a precise time schedule, raising the hypothesis that the cargo STAU2 delivers into IPCs changes over time. To test this, we combine RNA-immunoprecipitation with sequencing (RIP-seq) over four stages of mouse cortical development, generating a comprehensive cargo profile for STAU2. A subset of the cargo was 'stable', present at all stages, and involved in chromosome organization, macromolecule localization, translation and DNA repair. Another subset was 'dynamic', changing with cortical stage, and involved in neurogenesis, cell projection organization, neurite outgrowth, and included cortical layer markers. Notably, the dynamic STAU2 cargo included determinants of IPC versus neuronal fates and genes contributing to abnormal corticogenesis. Knockdown of one STAU2 target, Taf13, previously linked to microcephaly and impaired myelination, reduced oligodendrogenesis in vitro. We conclude that STAU2 contributes to the timing of corticogenesis by binding and delivering complex and temporally regulated RNA cargo into IPCs.

HuR counteracts miR-330 to promote STAT3 translation during inflammation-induced muscle wasting.

Debilitating cancer-induced muscle wasting, a syndrome known as cachexia, is lethal. Here we report a posttranscriptional pathway involving the RNA-binding protein HuR as a key player in the onset of this syndrome. Under these conditions, HuR switches its function from a promoter of muscle fiber formation to become an inducer of muscle loss. HuR binds to the STAT3 (signal transducer and activator of transcription 3) mRNA, which encodes one of the main effectors of this condition, promoting its expression both in vitro and in vivo. While HuR does not affect the stability and the cellular movement of this transcript, HuR promotes the translation of the STAT3 mRNA by preventing miR-330 (microRNA 330)-mediated translation inhibition. To achieve this effect, HuR directly binds to a U-rich element in the STAT3 mRNA-3'untranslated region (UTR) located within the vicinity of the miR-330 seed element. Even though the binding sites of HuR and miR-330 do not overlap, the recruitment of either one of them to the STAT3-3'UTR negatively impacts the binding and the function of the other factor. Therefore, together, our data establish the competitive interplay between HuR and miR-330 as a mechanism via which muscle fibers modulate, in part, STAT3 expression to determine their fate in response to promoters of muscle wasting.

KRAB-ZFP Repressors Enforce Quiescence of Oncogenic Human Herpesviruses.

Cancer-causing herpesviruses infect nearly every human and persist indefinitely in B lymphocytes in a quiescent state known as latency. A hallmark of this quiescence or latency is the presence of extrachromosomal viral genomes with highly restricted expression of viral genes. Silencing of viral genes ensures both immune evasion by the virus and limited pathology to the host, yet how multiple genes on multiple copies of viral genomes are simultaneously silenced is a mystery. In a unifying theme, we report that both cancer-causing human herpesviruses, despite having evolved independently, are silenced through the activities of two members of the Krüppel-associated box (KRAB) domain-zinc finger protein (ZFP) (KRAB-ZFP) epigenetic silencing family, revealing a novel STAT3-KRAB-ZFP axis of virus latency. This dual-edged antiviral strategy restricts the destructive ability of the lytic phase while promoting the cancer-causing latent phase. These findings also unveil roles for KRAB-ZFPs in silencing of multicopy foreign genomes with the promise of evicting herpesviruses to kill viral cancers bearing clonal viral episomes.IMPORTANCE Despite robust immune responses, cancer-causing viruses Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV) persist for life. This persistence is accomplished partly through a stealth mechanism that keeps extrachromosomal viral genomes quiescent. Quiescence, or latency, ensures that not every cell harboring viral genomes is killed directly through lytic activation or indirectly via the immune response, thereby evicting virus from host. For the host, quiescence limits pathology. Thus, both virus and host benefit from quiescence, yet how quiescence is maintained through silencing of a large set of viral genes on multiple viral genomes is not well understood. Our studies reveal that members of a gene-silencing family, the KRAB-ZFPs, promote quiescence of both cancer-causing human viruses through simultaneous silencing of multiple genes on multicopy extrachromosomal viral genomes.

FASTmiR: an RNA-based sensor for in vitro quantification and live-cell localization of small RNAs.

Small RNAs, including microRNAs (miRNAs) and small interfering RNAs (siRNAs), play a variety of important regulatory roles in many eukaryotes. Their small size has made it challenging to study them directly in live cells. Here we describe an RNA-based fluorescent sensor for small RNA detection both in vitro and in vivo, adaptable for any small RNA. It utilizes an sxRNA switch for detection of miRNA-mRNA interactions combined with a fluorophore-binding sequence 'Spinach', a GFP-like RNA aptamer for which the RNA-fluorophore complex exhibits strong and consistent fluorescence under an excitation wavelength. Two example sensors, FASTmiR171 and FASTmiR122, can rapidly detect and quantify the levels of miR171 and miR122 in vitro. The sensors can determine relative levels of miRNAs in total RNA extracts with sensitivity similar to small RNA sequencing and northern blots. FASTmiR sensors were also used to estimate the copy number range of miRNAs in total RNA extracts. To localize and analyze the spatial distribution of small RNAs in live, single cells, tandem copies of FASTmiR122 were expressed in different cell lines. FASTmiR122 was able to quantitatively detect the differences in miR122 levels in Huh7 and HEK293T cells demonstrating its potential for tracking miRNA expression and localization in vivo.

Integrative genomics positions MKRN1 as a novel ribonucleoprotein within the embryonic stem cell gene regulatory network.

In embryonic stem cells (ESCs), gene regulatory networks (GRNs) coordinate gene expression to maintain ESC identity; however, the complete repertoire of factors regulating the ESC state is not fully understood. Our previous temporal microarray analysis of ESC commitment identified the E3 ubiquitin ligase protein Makorin-1 (MKRN1) as a potential novel component of the ESC GRN. Here, using multilayered systems-level analyses, we compiled a MKRN1-centered interactome in undifferentiated ESCs at the proteomic and ribonomic level. Proteomic analyses in undifferentiated ESCs revealed that MKRN1 associates with RNA-binding proteins, and ensuing RIP-chip analysis determined that MKRN1 associates with mRNAs encoding functionally related proteins including proteins that function during cellular stress. Subsequent biological validation identified MKRN1 as a novel stress granule-resident protein, although MKRN1 is not required for stress granule formation, or survival of unstressed ESCs. Thus, our unbiased systems-level analyses support a role for the E3 ligase MKRN1 as a ribonucleoprotein within the ESC GRN.

Gene- and genome-based analysis of significant codon patterns in yeast, rat and mice genomes with the CUT Codon UTilization tool.

The translation of mRNA in all forms of life uses a three-nucleotide codon and aminoacyl-tRNAs to synthesize a protein. There are 64 possible codons in the genetic code, with codons for the ∼20 amino acids and 3 stop codons having 1- to 6-fold degeneracy. Recent studies have shown that families of stress response transcripts, termed modification tunable transcripts (MoTTs), use distinct codon biases that match specifically modified tRNAs to regulate their translation during a stress. Similarly, translational reprogramming of the UGA stop codon to generate selenoproteins or to perform programmed translational read-through (PTR) that results in a longer protein, requires distinct codon bias (i.e., more than one stop codon) and, in the case of selenoproteins, a specifically modified tRNA. In an effort to identify transcripts that have codon usage patterns that could be subject to translational control mechanisms, we have used existing genome and transcript data to develop the gene-specific Codon UTilization (CUT) tool and database, which details all 1-, 2-, 3-, 4- and 5-codon combinations for all genes or transcripts in yeast (Saccharomyces cerevisiae), mice (Mus musculus) and rats (Rattus norvegicus). Here, we describe the use of the CUT tool and database to characterize significant codon usage patterns in specific genes and groups of genes. In yeast, we demonstrate how the CUT database can be used to identify genes that have runs of specific codons (e.g., AGA, GAA, AAG) linked to translational regulation by tRNA methyltransferase 9 (Trm9). We further demonstrate how groups of genes can be analyzed to find significant dicodon patterns, with the 80 Gcn4-regulated transcripts significantly (P<0.00001) over-represented with the AGA-GAA dicodon. We have also used the CUT database to identify mouse and rat transcripts with internal UGA codons, with the surprising finding of 45 and 120 such transcripts, respectively, which is much larger than expected. The UGA data suggest that there could be many more translationally reprogrammed transcripts than currently reported. CUT thus represents a multi-species codon-counting database that can be used with mRNA-, translation- and proteomics-based results to better understand and model translational control mechanisms.

Identification of SMG6 cleavage sites and a preferred RNA cleavage motif by global analysis of endogenous NMD targets in human cells.

In metazoans, cleavage by the endoribonuclease SMG6 is often the first degradative event in non-sense-mediated mRNA decay (NMD). However, the exact sites of SMG6 cleavage have yet to be determined for any endogenous targets, and most evidence as to the identity of SMG6 substrates is indirect. Here, we use Parallel Analysis of RNA Ends to specifically identify the 5' termini of decay intermediates whose production is dependent on SMG6 and the universal NMD factor UPF1. In this manner, the SMG6 cleavage sites in hundreds of endogenous NMD targets in human cells have been mapped at high resolution. In addition, a preferred sequence motif spanning most SMG6 cleavage sites has been discovered and validated by mutational analysis. For many SMG6 substrates, depletion of SMG6 resulted in the accumulation of decapped transcripts, an effect indicative of competition between SMG6-dependent and SMG6-independent NMD pathways. These findings provide key insights into the mechanisms by which mRNAs targeted by NMD are degraded.

Profiling post-transcriptionally networked mRNA subsets using RIP-Chip and RIP-Seq.

Post-transcriptional regulation of messenger RNA contributes to numerous aspects of gene expression. The key component to this level of regulation is the interaction of RNA-binding proteins (RBPs) and their associated target mRNA. Splicing, stability, localization, translational efficiency, and alternate codon use are just some of the post-transcriptional processes regulated by RBPs. Central to our understanding of these processes is the need to characterize the network of RBP-mRNA associations and create a map of this functional post-transcriptional regulatory system. Here we provide a detailed methodology for mRNA isolation using RBP immunoprecipitation (RIP) as a primary partitioning approach followed by microarray (Chip) or next generation sequencing (NGS) analysis. We do this by using specific antibodies to target RBPs for the capture of associated RNA cargo. RIP-Chip/Seq has proven to be is a versatile, genomic technique that has been widely used to study endogenous RBP-RNA associations.

Combining temperature and force to study folding of an RNA hairpin.

RNA folding in cells typically occurs at mesophilic temperatures. However, in vitro, RNA can be unfolded either by increasing temperature to values that are much higher than physiological, or by mechanically pulling structures apart at ambient temperature. To directly study RNA folding at physiological temperatures and to unify thermodynamics measured by melting and pulling, we developed temperature-controlled optical tweezers (thermal tweezers) that can be used to mechanically unfold single RNA molecules at mesophilic temperatures. Folding of a 20-base-pair tetraloop hairpin was studied under different ionic conditions and at temperatures ranging from 22 °C to 42 °C. At each temperature, single hairpin molecules were held at constant force, and their two-state folding equilibria were monitored. The change in free energy derived from these measurements was used to construct a phase diagram of RNA structure using force and temperature as variables. Furthermore, we derived ΔG(0pN,T), the folding free energy at zero force and temperature T, by subtracting the stretching energy of unfolded RNA from the reversible mechanical work done to unfold the hairpin. ΔG(0pN,T) and its salt dependence agree reasonably well with the predictions by the nearest neighbor model. Under each ionic condition, ΔG(0pN,T) depended linearly on temperature, yielding ΔH(exp) and ΔS(exp) that also matched the predictions. The combination of force and temperature to study RNA folding is a step toward unifying thermodynamics measured by thermal melting and mechanical unfolding, and opens a new path for directly monitoring temperature induced RNA structural changes, as it occurs often in biology.

An in-depth map of polyadenylation sites in cancer.

We present a comprehensive map of over 1 million polyadenylation sites and quantify their usage in major cancers and tumor cell lines using direct RNA sequencing. We built the Expression and Polyadenylation Database to enable the visualization of the polyadenylation maps in various cancers and to facilitate the discovery of novel genes and gene isoforms that are potentially important to tumorigenesis. Analyses of polyadenylation sites indicate that a large fraction (∼30%) of mRNAs contain alternative polyadenylation sites in their 3' untranslated regions, independent of the cell type. The shortest 3' untranslated region isoforms are preferentially upregulated in cancer tissues, genome-wide. Candidate targets of alternative polyadenylation-mediated upregulation of short isoforms include POLR2K, and signaling cascades of cell-cell and cell-extracellular matrix contact, particularly involving regulators of Rho GTPases. Polyadenylation maps also helped to improve 3' untranslated region annotations and identify candidate regulatory marks such as sequence motifs, H3K36Me3 and Pabpc1 that are isoform dependent and occur in a position-specific manner. In summary, these results highlight the need to go beyond monitoring only the cumulative transcript levels for a gene, to separately analysing the expression of its RNA isoforms.

The essential role of stacking adenines in a two-base-pair RNA kissing complex.

In minimal RNA kissing complexes formed between hairpins with cognate GACG tetraloops, the two tertiary GC pairs are likely stabilized by the stacking of 5'-unpaired adenines at each end of the short helix. To test this hypothesis, we mutated the flanking adenines to various nucleosides and examined their effects on the kissing interaction. Electrospray ionization mass spectrometry was used to detect kissing dimers in a multiequilibria mixture, whereas optical tweezers were applied to monitor the (un)folding trajectories of single RNA molecules. The experimental findings were rationalized by molecular dynamics simulations. Together, the results showed that the stacked adenines are indispensable for the tertiary interaction. By shielding the tertiary base pairs from solvent and reducing their fraying, the stacked adenines made terminal pairs act more like interior base pairs. The purine double-ring of adenine was essential for effective stacking, whereas additional functional groups modulated the stabilizing effects through varying hydrophobic and electrostatic forces. Furthermore, formation of the kissing complex was dominated by base pairing, whereas its dissociation was significantly influenced by the flanking bases. Together, these findings indicate that unpaired flanking nucleotides play essential roles in the formation of otherwise unstable two-base-pair RNA tertiary interactions.

Transgenic expression of ZBP1 in neurons suppresses cocaine-associated conditioning.

To directly address whether regulating mRNA localization can influence animal behavior, we created transgenic mice that conditionally express Zipcode Binding Protein 1 (ZBP1) in a subset of neurons in the brain. ZBP1 is an RNA-binding protein that regulates the localization, as well as translation and stability of target mRNAs in the cytoplasm. We took advantage of the absence of ZBP1 expression in the mature brain to examine the effect of expressing ZBP1 on animal behavior. We constructed a transgene conditionally expressing a GFP-ZBP1 fusion protein in a subset of forebrain neurons and compared cocaine-cued place conditioning in these mice versus noninduced littermates. Transgenic ZBP1 expression resulted in impaired place conditioning relative to nonexpressing littermates, and acutely repressing expression of the transgene restored normal cocaine conditioning. To gain insight into the molecular changes that accounted for this change in behavior, we identified mRNAs that specifically immunoprecipitated with transgenic ZBP1 protein from the brains of these mice. These data suggest that RNA-binding proteins can be used as a tool to identify the post-transcriptional regulation of gene expression in the establishment and function of neural circuits involved in addiction behaviors.

PTSelect™: A post-transcriptional technology that enables rapid establishment of stable CHO cell lines and surveillance of clonal variation.

Currently, stable Chinese hamster ovary cell lines producing therapeutic, recombinant proteins are established either by antibiotic and/or metabolic selection. Here, we report a novel technology, PTSelect™ that utilizes an siRNA cloned upstream of the gene of interest (GOI) that is processed to produce functional PTSelect™-siRNAs, which enable cell enrichment. Cells with stably integrated GOI are selected and separated from cells without GOI by transfecting CD4/siRNA mRNA regulated by PTSelect™-siRNAs and exploiting the variable expression of CD4 on the cell surface. This study describes the PTSelect™ principle and compares the productivity, doubling time and stability of clones developed by PTSelect™ with conventionally developed clones. PTSelect™ rapidly established a pool population with comparable stability and productivity to pools generated by traditional methods and can further be used to easily monitor productivity changes due to clonal drift, identifying individual cells with reduced productivity.

Redox and mTOR-dependent regulation of plasma lamellar calcium influx controls the senescence-associated secretory phenotype.

IMPACT STATEMENT: Through its ability to evoke responses from cells in a paracrine fashion, the senescence-associated secretory phenotype (SASP) has been linked to numerous age-associated disease pathologies including tumor invasion, cardiovascular dysfunction, neuroinflammation, osteoarthritis, and renal disease. Strategies which limit the amplitude and duration of SASP serve to delay age-related degenerative decline. Here we demonstrate that the SASP regulation is linked to shifts in intracellular Ca2+ homeostasis and strategies which rescue redox-dependent calcium entry including enzymatic H2O2 scavenging, TRP modulation, or mTOR inhibition block SASP and TRPC6 gene expression. As Ca2+ is indispensable for secretion from both secretory and non-secretory cells, it is exciting to speculate that the expression of plasma lamellar TRP channels critical for the maintenance of intracellular Ca2+ homeostasis may be coordinately regulated with the SASP.

Informatic resources for identifying and annotating structural RNA motifs.

Post-transcriptional regulation of genes and transcripts is a vital aspect of cellular processes, and unlike transcriptional regulation, remains a largely unexplored domain. One of the most obvious and most important questions to explore is the discovery of functional RNA elements. Many RNA elements have been characterized to date ranging from cis-regulatory motifs within mRNAs to large families of non-coding RNAs. Like protein coding genes, the functional motifs of these RNA elements are highly conserved, but unlike protein coding genes, it is most often the structure and not the sequence that is conserved. Proper characterization of these structural RNA motifs is both the key and the limiting step to understanding the post-transcriptional aspects of the genomic world. Here, we focus on the task of structural motif discovery and provide a survey of the informatics resources geared towards this task.

Surface Enhanced Raman Spectroscopy for Single Molecule Protein Detection.

A two-step process of protein detection at a single molecule level using SERS was developed as a proof-of-concept platform for medical diagnostics. First, a protein molecule was bound to a linker in the bulk solution and then this adduct was chemically reacted with the SERS substrate. Traut's Reagent (TR) was used to thiolate Bovine serum albumin (BSA) in solution followed by chemical cross linking to a gold surface through a sulfhydryl group. A Glycine-TR adduct was used as a control sample to identify the protein contribution to the SER spectra. Gold SERS substrates were manufactured by electrochemical deposition. Solutions at an ultralow concentration were used for attaching the TR adducts to the SERS substrate. Samples showed the typical behavior of a single molecule SERS including spectral fluctuations, blinking and Raman signal being generated from only selected points on the substrate. The fluctuating SER spectra were examined using Principle Component Analysis. This unsupervised statistics allowed for the selecting of spectral contribution from protein moiety indicating that the method was capable of detecting a single protein molecule. Thus we have demonstrated, that the developed two-step methodology has the potential as a new platform for medical diagnostics.

NF-kappa B-mediated MyoD decay during muscle wasting requires nitric oxide synthase mRNA stabilization, HuR protein, and nitric oxide release.

Muscle wasting (cachexia) is a consequence of chronic diseases, such as cancer, and is associated with degradation of muscle proteins such as MyoD. The cytokines tumor necrosis factor alpha and gamma interferon induce muscle degeneration by activating the transcription factor NF-kappaB and its target genes. Here, we show that a downstream target of NF-kappaB is the nitric oxide (NO) synthase gene (iNos) and suggest that NO production stimulates MyoD mRNA loss. In fact, although cytokine treatment of iNos(-/-) mice activated NF-kappaB, it did not trigger MyoD mRNA degeneration, demonstrating that NF-kappaB-mediated muscle wasting requires an active iNOS-NO pathway. The induced expression of iNOS by cytokines relies on both transcriptional activation via NF-kappaB and increased mRNA stability via the RNA-binding protein HuR. Moreover, we show that HuR regulates iNOS expression in an AMP-activated protein kinase (AMPK)-dependent manner. Furthermore, AMPK activation results in HuR nuclear sequestration, inhibition of iNOS synthesis, and reduction in cytokine-induced MyoD loss. These results define iNOS and HuR as critical players in cytokine-induced cachexia, establishing them as potential therapeutic targets.

Advances in RIP-chip analysis : RNA-binding protein immunoprecipitation-microarray profiling.

In eukaryotic organisms, gene regulatory networks require an additional level of coordination that links transcriptional and post-transcriptional processes. Messenger RNAs have traditionally been viewed as passive molecules in the pathway from transcription to translation. However, it is now clear that RNA-binding proteins (RBPs) play a major role in regulating multiple mRNAs to facilitate gene expression patterns. On this basis, post-transcriptional and transcriptional gene expression networks appear to be very analogous. Our previous research focused on targeting RBPs to develop a better understanding of post-transcriptional gene-expression processing and the regulation of mRNA networks. We developed technologies for purifying endogenously formed RBP-mRNA complexes from cellular extracts and identifying the associated messages using genome-scale, microarray technology, a method called ribonomics or RNA-binding protein immunoprecipitation-microarray (Chip) profiling or RIP-Chip. The use of the RIP-Chip methods has provided great insight into the infrastructure of coordinated eukaryotic post-transcriptional gene expression, insights which could not have been obtained using traditional RNA expression profiling approaches (1). This chapter describes the most current RIP-Chip techniques as we presently practice them. We also discuss some of the informatic aspects that are unique to analyzing RIP-Chip data.

Bioinformatic tools for studying post-transcriptional gene regulation : The UAlbany TUTR collection and other informatic resources.

The untranslated regions (UTRs) of many mRNAs contain sequence and structural motifs that are used to regulate the stability, localization, and translatability of the mRNA. It should be possible to discover previously unidentified RNA regulatory motifs by examining many related nucleotide sequences, which are assumed to contain a common motif. This is a general practice for discovery of DNA-based sequence-based patterns, in which alignment tools are heavily exploited. However, because of the complexity of sequential and structural components of RNA-based motifs, simple-alignment tools are frequently inadequate. The consensus sequences they find frequently have the potential for significant variability at any given position and are only loosely characterized. The development of RNA-motif discovery tools that infer and integrate structural information into motif discovery is both necessary and expedient. Here, we provide a selected list of existing web-accessible algorithms for the discovery of RNA motifs, which, although not exhaustive, represents the current state of the art. To facilitate the development, evaluation, and training of new software programs that identify RNA motifs, we created the UAlbany training UTR (TUTR) database, which is a collection of validated sets of sequences containing experimentally defined regulatory motifs. Presently, eleven training sets have been generated with associated indexes and "answer sets" provided that identify where the previously characterized RNA motif [the iron responsive element (IRE), AU-rich class-2 element (ARE), selenocysteine insertion sequence (SECIS), etc.] resides in each sequence. The UAlbany TUTR collection is a shared resource that is available to researchers for software development and as a research aid.