Human immunodeficiency virus type 1 (HIV-1) replication requires the expression of two classes of viral mRNA. The early class of HIV-1 transcripts is fully spliced and encodes viral regulatory gene products. The functional expression of one of these nuclear regulatory proteins, termed Rev (formerly Art or Trs), induces the cytoplasmic expression of the incompletely spliced, late class of HIV-1 mRNAs that encode the viral structural proteins, including Gag and Env. Here, we provide evidence that this induction reflects the export from the cell nucleus to the cytoplasm of a pool of unspliced viral RNA constitutively expressed in the nucleus. The hypothesis that Rev acts on RNA transport, rather than splicing, is further supported by the observation that the cytoplasmic expression of a non-spliceable HIV-1 env gene sequence is also subject to Rev regulation. Here we show that this Rev response requires a specific target sequence which coincides with a complex RNA secondary structure present in the env gene. The response to Rev is fully maintained when this sequence is relocated to other exonic or intronic locations within env but is ablated by inversion. These results indicate that the HIV-1 rev gene product induces HIV-1 structural gene expression by activating the sequence-specific nuclear export of incompletely spliced HIV-1 RNA species.

We provide a large-scale dataset on absolute protein and matching mRNA concentrations from the human medulloblastoma cell line Daoy. The correlation between mRNA and protein concentrations is significant and positive (Rs=0.46, R2=0.29, P-value<2e16), although non-linear.Out of ∼200 tested sequence features, sequence length, frequency and properties of amino acids, as well as translation initiation-related features are the strongest individual correlates of protein abundance when accounting for variation in mRNA concentration.When integrating mRNA expression data and all sequence features into a non-parametric regression model (Multivariate Adaptive Regression Splines), we were able to explain up to 67% of the variation in protein concentrations. Half of the contributions were attributed to mRNA concentrations, the other half to sequence features relating to regulation of translation and protein degradation. The sequence features are primarily linked to the coding and 3′ untranslated region. To our knowledge, this is the most comprehensive predictive model of human protein concentrations achieved so far.

Transient receptor potential melastatin member 8 (TRPM8) is a calcium ion (Ca2+)–permeable cation channel that serves as the primary cold and menthol sensor in humans. Activation of TRPM8 by cooling compounds relies on allosteric actions of agonist and membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP2), but lack of structural information has thus far precluded a mechanistic understanding of ligand and lipid sensing by TRPM8. Using cryo–electron microscopy, we determined the structures of TRPM8 in complex with the synthetic cooling compound icilin, PIP2, and Ca2+, as well as in complex with the menthol analog WS-12 and PIP2. Our structures reveal the binding sites for cooling agonists and PIP2in TRPM8. Notably, PIP2binds to TRPM8 in two different modes, which illustrate the mechanism of allosteric coupling between PIP2and agonists. This study provides a platform for understanding the molecular mechanism of TRPM8 activation by cooling agents.

In plants and invertebrate animals, RNA silencing is a form of nucleic acid-based adaptive immunity. By contrast, jawed vertebrates have evolved complex protein-based adaptive immunity. Although short interfering RNAs (siRNAs) have been used as artificial tools to silence viral infection in human cells, it remains unknown whether mammalian viruses naturally elicit such immunity in vertebral cells. Here, we report the evidence that HIV-1 encodes viral siRNA precursors in its genome and that natural HIV-1 infection provokes nucleic acid-based immunity in human cells. To combat this cellular defense, HIV-1 has evolved in its Tat protein a suppressor of RNA silencing (SRS) function. Tat abrogates the cell's RNA-silencing defense by subverting the ability of Dicer to process precursor double-stranded RNAs into siRNAs.

Small non-coding RNAs of 18–25 nt in length can regulate gene expression through the RNA interference (RNAi) pathway. To characterize small RNAs in HIV-1-infected cells, we performed linker-ligated cloning followed by high-throughput pyrosequencing. Here, we report the composition of small RNAs in HIV-1 productively infected MT4 T-cells. We identified several HIV-1 small RNA clones and a highly abundant small 18-nt RNA that is antisense to the HIV-1 primer-binding site (PBS). This 18-nt RNA apparently originated from the dsRNA hybrid formed by the HIV-1 PBS and the 3′ end of the human cellular tRNAlys3. It was found to associate with the Ago2 protein, suggesting its possible function in the cellular RNAi machinery for targeting HIV-1.

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Kaposi sarcoma-associated herpesvirus (KSHV) ORF57, also known as Mta (mRNA transcript accumulation), enhances viral intron-less transcript accumulation and promotes splicing of intron-containing viral RNA transcripts. In this study, we identified KSHV PAN, a long non-coding polyadenylated nuclear RNA as a main target of ORF57 by a genome-wide CLIP (cross-linking and immunoprecipitation) approach. KSHV genome lacking ORF57 expresses only a minimal amount of PAN. In cotransfection experiments, ORF57 alone increased PAN expression by 20-30-fold when compared to vector control. This accumulation function of ORF57 was dependent on a structured RNA element in the 5' PAN, named MRE (Mta responsive element), but not much so on an ENE (expression and nuclear retention element) in the 3' PAN previously reported by other studies. We showed that the major function of the 5' PAN MRE is increasing the RNA half-life of PAN in the presence of ORF57. Further mutational analyses revealed a core motif consisting of 9 nucleotides in the MRE-II , which is responsible for ORF57 interaction and function. The 9-nt core in the MRE-II also binds cellular PABPC1, but not the E1B-AP5 which binds another region of the MRE-II. In addition, we found that PAN RNA is partially exportable in the presence of ORF57. Together, our data provide compelling evidence as to how ORF57 functions to accumulate a non-coding viral RNA in the course of virus lytic infection.

Immunity 22, 607-619; May 2005) On page 618 in the siRNA and shRNA Constructions section of our article, the primer for vsiRNA1 was misprinted due to a typographical error. The correct primer for this should be (5#-TCCTTGGGTTCTTGGGAGCTTCAAGAGAGCTCC TAAGAACCCAAGGATTTTTT-3#).