Chia-Ying Chu

Translation repression in human cells by microRNA-induced gene silencing requires RCK/p54.

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.

Small RNAs: Regulators and guardians of the genome

Small non‐coding RNAs comprise several classes and sizes, but all share a unifying function in cellular physiology: epigenetic regulation of gene expression. Here, we review the salient aspects of recent studies on the biogenesis and function of three classes of small RNAs: miRNAs, siRNAs, and piRNAs. Although the mechanisms are becoming clear by which siRNA‐triggered mRNA cleavage silences genes, more studies are needed on several issues regarding miRNA‐mediated translation repression. Piwi proteins have been suggested to co‐operate in amplifying piRNA biogenesis to maintain transposon silencing in the germ line genome, but details of this process are still unknown as well as the functional consequences of piRNA expression at discrete genomic loci. J. Cell. Physiol. 213: 412–419, 2007.

Target accessibility dictates the potency of human RISC

In this report, we examined the effect of increased target site access on activated human RNA-induced silencing complex (RISC*) catalysis. Kinetic studies revealed that siRNA-programmed RISC* cleaved target RNA with higher efficiencies when target site access was increased. These results provide evidence that target site access is linked to RISC* catalysis.

MicroRNAs encoded by Kaposi's sarcoma-associated herpesvirus regulate viral life cycle

Kaposis sarcoma‐associated herpesvirus (KSHV) is linked with Kaposis sarcoma and lymphomas. The pathogenesis of KSHV depends on the balance between two phases of the viral cycle: latency and lytic replication. In this study, we report that KSHV‐encoded microRNAs (miRNAs) function as regulators by maintaining viral latency and inhibiting viral lytic replication. MiRNAs are short, noncoding, small RNAs that post‐transcriptionally regulate the expression of messenger RNAs. Of the 12 viral miRNAs expressed in latent KSHV‐infected cells, we observed that expression of miR‐K3 can suppress both viral lytic replication and gene expression. Further experiments indicate that miR‐K3 can regulate viral latency by targeting nuclear factor I/B. Nuclear factor I/B can activate the promoter of the viral immediate‐early transactivator replication and transcription activator (RTA), and depletion of nuclear factor I/B by short hairpin RNAs had similar effects on the viral life cycle to those of miR‐K3. Our results suggest a role for KSHV miRNAs in regulating the viral life cycle.

LIM-domain proteins, LIMD1, Ajuba, and WTIP are required for microRNA-mediated gene silencing

In recent years there have been major advances with respect to the identification of the protein components and mechanisms of microRNA (miRNA) mediated silencing. However, the complete and precise repertoire of components and mechanism(s) of action remain to be fully elucidated. Herein we reveal the identification of a family of three LIM domain-containing proteins, LIMD1, Ajuba and WTIP (Ajuba LIM proteins) as novel mammalian processing body (P-body) components, which highlight a novel mechanism of miRNA-mediated gene silencing. Furthermore, we reveal that LIMD1, Ajuba, and WTIP bind to Ago1/2, RCK, Dcp2, and eIF4E in vivo, that they are required for miRNA-mediated, but not siRNA-mediated gene silencing and that all three proteins bind to the mRNA 5′ m7GTP cap–protein complex. Mechanistically, we propose the Ajuba LIM proteins interact with the m7GTP cap structure via a specific interaction with eIF4E that prevents 4EBP1 and eIF4G interaction. In addition, these LIM-domain proteins facilitate miRNA-mediated gene silencing by acting as an essential molecular link between the translationally inhibited eIF4E-m7GTP-5′cap and Ago1/2 within the miRISC complex attached to the 3′-UTR of mRNA, creating an inhibitory closed-loop complex.

The new face of the old molecules: crustin Pm4 and transglutaminase type I serving as rnps down-regulate astakine-mediated hematopoiesis.

Astakine is an important cytokine that is involved in crustacean hematopoiesis. Interestingly, the protein levels of astakine increased dramatically in plasma of LPS-injected shrimp while mRNA levels remained unchanged. Here, we investigated the involvement of astakine 3′-untranslated region (UTR) in its protein expression. The 3′-UTR of astakine down-regulated the expression of reporter protein but the mRNA stability of reporter gene was unaffected. We identified the functional regulatory elements of astakine 3′-UTR, where 3′-UTR242–483 acted as repressor. The electrophoresis mobility shift assay (EMSA), RNA pull-down assay and LC/MS/MS were performed to identify the protein association. We noted that crustin Pm4 and shrimp transglutaminase I (STG I) were associated to astakine 3′-UTR242–483, while two other proteins have yet to be revealed. Depletion of hemocytic crustin Pm4 and STG I significantly increased the protein level of astakine while astakine mRNA level remained unaffected. Lipopolysaccharide (LPS) stimulated the secretion of crustin Pm4 and STG I from hemocytes to plasma and increased the astakine level to stimulate the hemocytes proliferation. Altogether, we identified the shrimp crustin Pm4 and STG I as novel RNA binding proteins that play an important role in down-regulating astakine expression at post-transcriptional level and are crucial for the maintenance of hematopoiesis.

Dual mechanisms regulate the nucleocytoplasmic localization of human DDX6

DDX6 is a conserved DEAD-box protein (DBP) that plays central roles in cytoplasmic RNA regulation, including processing body (P-body) assembly, mRNA decapping, and translational repression. Beyond its cytoplasmic functions, DDX6 may also have nuclear functions because its orthologues are known to localize to nuclei in several biological contexts. However, it is unclear whether DDX6 is generally present in human cell nuclei, and the molecular mechanism underlying DDX6 subcellular distribution remains elusive. In this study, we showed that DDX6 is commonly present in the nuclei of human-derived cells. Our structural and molecular analyses deviate from the current model that the shuttling of DDX6 is directly mediated by the canonical nuclear localization signal (NLS) and nuclear export signal (NES), which are recognized and transported by Importin-α/β and CRM1, respectively. Instead, we show that DDX6 can be transported by 4E-T in a piggyback manner. Furthermore, we provide evidence for a novel nuclear targeting mechanism in which DDX6 enters the newly formed nuclei by “hitch-hiking” on mitotic chromosomes with its C-terminal domain during M phase progression. Together, our results indicate that the nucleocytoplasmic localization of DDX6 is regulated by these dual mechanisms.

Shrimp miR-10a is co-opted by white spot syndrome virus to increase viral gene expression and viral replication

Members of the microRNA miR-10 family are highly conserved and play many important roles in diverse biological mechanisms, including immune-related responses and cancer-related processes in certain types of cancer. In this study, we found the most highly upregulated shrimp microRNA from Penaeus vannamei during white spot syndrome virus (WSSV) infection was miR-10a. After confirming the expression level of miR-10a by northern blot and quantitative RT-PCR, an in vivo experiment showed that the viral copy number was decreased in miR-10a-inhibited shrimp. We found that miR-10a targeted the 5′ untranslated region (UTR) of at least three viral genes (vp26, vp28, and wssv102), and plasmids that were controlled by the 5′ UTR of these genes produced enhanced luciferase signals in transfected SF9 cells. These results suggest a previously unreported role for shrimp miR-10a and even a new type of host–virus interaction, whereby a co-opts the key cellular regulator miR-10a to globally enhance the translation of viral proteins.

Structure and Gene-Silencing Mechanisms of Small Noncoding RNAs

Small (19–31-nucleotides) noncoding RNAs were identified in the past 10 years for their distinct function in gene silencing. The best known gene-silencing phenomenon, RNA interference (RNAi), is triggered in a sequence-specific manner by endogenously produced or exogenously introduced small doubled-stranded RNAs. As knowledge of the structure and function of the RNAi machinery has expanded, this phenomenon has become a powerful tool for biochemical research; it has enormous potential for therapeutics. This chapter summarizes significant aspects of three major classes of small noncoding, regulatory RNAs: small interfering RNAs (siRNAs), microRNAs (miRNAs), and Piwi-interacting RNAs (piRNAs). Here, we focus on the biogenesis of these small RNAs, their structural features and coupled effectors as well as the mechanisms of each small regulatory RNA pathway which reveal fascinating ways by which gene silencing is controlled and fine-tuned at an epigenetic level.