RNA interference is triggered by double-stranded RNA that is processed into small interfering RNAs (siRNAs) by Dicer enzyme. Endogenously, RNA interference triggers are created from small noncoding RNAs called microRNAs (miRNAs). RNA-induced silencing complexes (RISC) in human cells can be programmed by exogenously introduced siRNA or endogenously expressed miRNA. siRNA-programmed RISC (siRISC) silences expression by cleaving a perfectly complementary target mRNA, whereas miRNA-induced silencing complexes (miRISC) inhibits translation by binding imperfectly matched sequences in the 3′ UTR of target mRNA. Both RISCs contain Argonaute2 (Ago2), which catalyzes target mRNA cleavage by siRISC and localizes to cytoplasmic mRNA processing bodies (P-bodies). Here, we show that RCK/p54, a DEAD box helicase, interacts with argonaute proteins, Ago1 and Ago2, in affinity-purified active siRISC or miRISC from human cells; directly interacts with Ago1 and Ago2 in vivo, facilitates formation of P-bodies, and is a general repressor of translation. Disrupting P-bodies by depleting Lsm1 did not affect RCK/p54 interactions with argonaute proteins and its function in miRNA-mediated translation repression. Depletion of RCK/p54 disrupted P-bodies and dispersed Ago2 throughout the cytoplasm but did not significantly affect siRNA-mediated RNA functions of RISC. Depleting RCK/p54 released general, miRNA-induced, and let-7-mediated translational repression. Therefore, we propose that translation repression is mediated by miRISC via RCK/p54 and its specificity is dictated by the miRNA sequence binding multiple copies of miRISC to complementary 3′ UTR sites in the target mRNA. These studies also suggest that translation suppression by miRISC does not require P-body structures, and location of miRISC to P-bodies is the consequence of translation repression.

SUMMARY MicroRNAs (miRNAs), ~22-nucleotide noncoding RNAs, assemble into RNA-induced silencing complexes (RISC) and localize to cytoplasmic substructures called P-bodies. Dictated by base-pair complementarity between miRNA and a target mRNA, miRNAs specifically repress posttranscriptional expression of several mRNAs. Here, we report that HIV-1 mRNA interacts with RISC proteins and that disrupting P-body structures enhances viral production and infectivity. In HIV-1-infected human T lymphocytes, we identified a highly abundant miRNA, miR-29a, which specifically targets the HIV-1 3’-UTR region. Inhibiting miR-29a enhanced HIV-1 viral production and infectivity, whereas expressing a miR-29 mimic suppressed viral replication. We also found that specific miR-29a-HIV-1 mRNA interactions enhance viral mRNA association with RISC and P-body proteins. Thus, we provide an example of a single host miRNA regulating HIV-1 production and infectivity. These studies highlight the significance of miRNAs and P-bodies in modulating host cell interactions with HIV-1 and possibly other viruses.

Due to the importance of protein phosphorylation in cellular control, many researches are undertaken to predict the kinase-specific phosphorylation sites. Referred to our previous work, KinasePhos 1.0, incorporated profile hidden Markov model (HMM) with flanking residues of the kinase-specific phosphorylation sites. Herein, a new web server, KinasePhos 2.0, incorporates support vector machines (SVM) with the protein sequence profile and protein coupling pattern, which is a novel feature used for identifying phosphorylation sites. The coupling pattern [XdZ] denotes the amino acid coupling-pattern of amino acid types X and Z that are separated by d amino acids. The differences or quotients of coupling strength CXdZ between the positive set of phosphorylation sites and the background set of whole protein sequences from Swiss-Prot are computed to determine the number of coupling patterns for training SVM models. After the evaluation based on k-fold cross-validation and Jackknife cross-validation, the average predictive accuracy of phosphorylated serine, threonine, tyrosine and histidine are 90, 93, 88 and 93%, respectively. KinasePhos 2.0 performs better than other tools previously developed. The proposed web server is freely available at http://KinasePhos2.mbc.nctu.edu.tw/.

RNA interference (RNAi) is the process by which short-interfering RNA (siRNA) target a specific mRNA for degradation through interactions with an RNA-induced silencing complex (RISC). Here, a clear correlation between siRNA localization, cellular uptake, and RNAi activity was discovered by delivering siRNA into cells using siRNA-TAT(47-57) peptide, siRNA-TAT(47-57)-derived oligocarbamate conjugates, or nanoparticles. For successful RNAi, the localization of siRNA was distinctly perinuclear, suggesting that siRNA is targeted to these regions for interactions with RISC to induce RNAi. siRNA sequence variation and the presence of the target mRNA apparently did not change the subcellular localization pattern of siRNA. Intriguingly, siRNA conjugated to TAT(47-57) peptide or TAT(47-57)-derived oligocarbamate resulted in efficient RNAi activity and perinuclear localization of siRNA that was distinctly different from nonconjugated free TAT peptide nucleolar localization. These results suggest that interactions with RISC dictate siRNA localization even when siRNA is conjugated to TAT(47-57) peptide.