The CRTC1-SIK1 pathway regulates entrainment of the circadian clock.

Retinal photoreceptors entrain the circadian system to the solar day. This photic resetting involves cAMP response element binding protein (CREB)-mediated upregulation of Per genes within individual cells of the suprachiasmatic nuclei (SCN). Our detailed understanding of this pathway is poor, and it remains unclear why entrainment to a new time zone takes several days. By analyzing the light-regulated transcriptome of the SCN, we have identified a key role for salt inducible kinase 1 (SIK1) and CREB-regulated transcription coactivator 1 (CRTC1) in clock re-setting. An entrainment stimulus causes CRTC1 to coactivate CREB, inducing the expression of Per1 and Sik1. SIK1 then inhibits further shifts of the clock by phosphorylation and deactivation of CRTC1. Knockdown of Sik1 within the SCN results in increased behavioral phase shifts and rapid re-entrainment following experimental jet lag. Thus SIK1 provides negative feedback, acting to suppress the effects of light on the clock. This pathway provides a potential target for the regulation of circadian rhythms.

Hepatocyte-targeted RNAi therapeutics for the treatment of chronic hepatitis B virus infection.

RNA interference (RNAi)-based therapeutics have the potential to treat chronic hepatitis B virus (HBV) infection in a fundamentally different manner than current therapies. Using RNAi, it is possible to knock down expression of viral RNAs including the pregenomic RNA from which the replicative intermediates are derived, thus reducing viral load, and the viral proteins that result in disease and impact the immune system's ability to eliminate the virus. We previously described the use of polymer-based Dynamic PolyConjugate (DPC) for the targeted delivery of siRNAs to hepatocytes. Here, we first show in proof-of-concept studies that simple coinjection of a hepatocyte-targeted, N-acetylgalactosamine-conjugated melittin-like peptide (NAG-MLP) with a liver-tropic cholesterol-conjugated siRNA (chol-siRNA) targeting coagulation factor VII (F7) results in efficient F7 knockdown in mice and nonhuman primates without changes in clinical chemistry or induction of cytokines. Using transient and transgenic mouse models of HBV infection, we show that a single coinjection of NAG-MLP with potent chol-siRNAs targeting conserved HBV sequences resulted in multilog repression of viral RNA, proteins, and viral DNA with long duration of effect. These results suggest that coinjection of NAG-MLP and chol-siHBVs holds great promise as a new therapeutic for patients chronically infected with HBV.

Treatment of the metabolic syndrome by siRNA targeting apolipoprotein CIII.

Apolipoprotein CIII (apoCIII) is increased in obesity-induced insulin resistance and type-2 diabetes. Emerging evidences support the advantages of small interfering RNAs (siRNAs) to target disease-causing genes. The aim of this study was to develop siRNAs for in vivo silencing of apoCIII and investigate if this results in metabolic improvements comparable to what we have seen using antisense oligonucelotides against apoCIII. Twenty-four siRNAs were synthesized and tested in a dual luciferase reporter assay. The eight best were selected, based on knockdown at 20 nM, and of these, two were selected based on IC50 values. In vivo experiments were performed in ob/ob mice, an obese animal model for diabetes. To determine the dose-dependency, efficacy, duration of effect and therapeutic dose we used a short protocol giving the apoCIII-siRNA mix for three days. To evaluate long-term metabolic effects mice were treated for three days, every second week for eight weeks. The siRNA mix effectively and selectively reduced expression of apoCIII in liver in vivo. Treatment had to be repeated every two weeks to maintain a suppression of apoCIII. The reduction of apoCIII resulted in increased LPL activity, lower triglycerides, reduced liver fat, ceased weight gain, enhanced insulin sensitivity, and improved glucose homeostasis. No off-target or side effects were observed during the eight-week treatment period. These results suggest that in vivo silencing of apoCIII with siRNA, is a promising approach with the potential to be used in the battle against obesity-induced metabolic disorders.

Identification of Novel Fibrosis Modifiers by In Vivo siRNA Silencing.

Fibrotic diseases contribute to 45% of deaths in the industrialized world, and therefore a better understanding of the pathophysiological mechanisms underlying tissue fibrosis is sorely needed. We aimed to identify novel modifiers of tissue fibrosis expressed by myofibroblasts and their progenitors in their disease microenvironment through RNA silencing in vivo. We leveraged novel biology, targeting genes upregulated during liver and kidney fibrosis in this cell lineage, and employed small interfering RNA (siRNA)-formulated lipid nanoparticles technology to silence these genes in carbon-tetrachloride-induced liver fibrosis in mice. We identified five genes, Egr2, Atp1a2, Fkbp10, Fstl1, and Has2, which modified fibrogenesis based on their silencing, resulting in reduced Col1a1 mRNA levels and collagen accumulation in the liver. These genes fell into different groups based on the effects of their silencing on a transcriptional mini-array and histological outcomes. Silencing of Egr2 had the broadest effects in vivo and also reduced fibrogenic gene expression in a human fibroblast cell line. Prior to our study, Egr2, Atp1a2, and Fkbp10 had not been functionally validated in fibrosis in vivo. Thus, our results provide a major advance over the existing knowledge of fibrogenic pathways. Our study is the first example of a targeted siRNA assay to identify novel fibrosis modifiers in vivo.

Gene silencing with siRNA duplexes composed of target-mRNA-complementary and partially palindromic or partially complementary single-stranded siRNAs.

Synthetic small interfering RNA (siRNA) duplexes are widely used to transiently and sequence-specifically disrupt gene expression in mammalian cultured cells. The efficiency and specificity of mRNA cleavage is partly affected by the presence of the nontargeting "passenger" or "sense" siRNA strand, which is required for presentation of the target-complementary or guide siRNA strand to the double-strand-specific RNA silencing protein machinery. We show that siRNA duplexes can be designed that are solely composed of two fully target-complementary guide strands that are sufficiently complementary to each other to form stable duplexes with characteristic 3' overhanging ends. The general feasibility of this approach is documented by transient knockdown of lamin A/C and emerin in HeLa cells. The silencing efficiencies of guide-only siRNA duplexes are comparable to prototypical fully paired passenger/guide duplex siRNAs, even though guide-only siRNA duplexes may contain a significant number of nonWatson-Crick and G/U wobble base pairs. Such siRNA duplexes may offer advantages regarding production costs and specificity of gene silencing.

siRNA Selection Server: an automated siRNA oligonucleotide prediction server.

The Whitehead siRNA (short interfering RNA) Selection Web Server (http://jura.wi.mit.edu/bioc/siRNA) automates the design of short oligonucleotides that can specifically 'knock down' expression of target genes. These short sequences are about 21 nt in length, and when synthesized as double stranded RNA and introduced into cell culture, can reduce or eliminate the function of the target gene. Depending on the length of a gene, there are potentially numerous combinations of possible 21mers. Some experimental evidence has already shown that not all 21mers in a gene have the same effectiveness at silencing gene function. Our tool incorporates published design rules and presents the scientist with information about uniqueness of the 21mers within the genome, thermodynamic stability of the double stranded RNA duplex, GC content, presence of SNPs and other features that may contribute to the effectiveness of a siRNA.

Hepatitis B virus genome replication triggers toll-like receptor 3-dependent interferon responses in the absence of hepatitis B surface antigen.

The hepatitis B virus (HBV) has been described as stealth virus subverting immune responses initially upon infection. Impaired toll-like receptor signaling by the HBV surface antigen (HBsAg) attenuates immune responses to facilitate chronic infection. This implies that HBV replication may trigger host innate immune responses in the absence of HBsAg. Here we tested this hypothesis, using highly replicative transgenic mouse models. An HBV replication-dependent expression of antiviral genes was exclusively induced in HBsAg-deficient mice. These interferon responses attributed to toll-like receptor 3 (TLR3)-activated Kupffer and liver sinusoidal endothelial cells and further controlled the HBV genome replication. However, activation of TLR3 with exogenous ligands indicated additional HBs-independent immune evasion events. Our data demonstrate that in the absence of HBsAg, hepatic HBV replication leads to Tlr3-dependent interferon responses in non-parenchymal liver cells. We hypothesize that HBsAg is a major HBV-mediated evasion mechanism controlling endogenous antiviral responses in the liver. Eradication of HBsAg as a therapeutic goal might facilitate the induction of endogenous antiviral immune responses in patients chronically infected with HBV.

Isoforms of Melanopsin Mediate Different Behavioral Responses to Light.

Melanopsin (OPN4) is a retinal photopigment that mediates a wide range of non-image-forming (NIF) responses to light including circadian entrainment, sleep induction, the pupillary light response (PLR), and negative masking of locomotor behavior (the acute suppression of activity in response to light). How these diverse NIF responses can all be mediated by a single photopigment has remained a mystery. We reasoned that the alternative splicing of melanopsin could provide the basis for functionally distinct photopigments arising from a single gene. The murine melanopsin gene is indeed alternatively spliced, producing two distinct isoforms, a short (OPN4S) and a long (OPN4L) isoform, which differ only in their C terminus tails. Significantly, both isoforms form fully functional photopigments. Here, we show that different isoforms of OPN4 mediate different behavioral responses to light. By using RNAi-mediated silencing of each isoform in vivo, we demonstrated that the short isoform (OPN4S) mediates light-induced pupillary constriction, the long isoform (OPN4L) regulates negative masking, and both isoforms contribute to phase-shifting circadian rhythms of locomotor behavior and light-mediated sleep induction. These findings demonstrate that splice variants of a single receptor gene can regulate strikingly different behaviors.

Identification, cloning, and functional analysis of the human U6 snRNA-specific terminal uridylyl transferase.

Mammalian cells contain a highly specific terminal uridylyl transferase (TUTase) that exclusively accepts U6 snRNA as substrate. This enzyme, termed U6-TUTase, was purified from HeLa cell extracts and analyzed by microsequencing. All sequenced peptides matched a unique human cDNA coding for a previously unknown protein. Domain structure analysis revealed that the U6-TUTase also belongs to the well-characterized poly(A) polymerase protein superfamily. However, by amino acid sequence as well as RNA-binding motifs, human U6-TUTase is highly divergent from both the poly(A) polymerases and from the TUTases identified within the editing complexes of trypanosomes. After cloning, the recombinant U6-TUTase was expressed in HeLa cells. Analysis of its catalytical activity confirmed the identity of the cloned protein as U6-TUTase, exhibiting the same exclusive substrate specificity for U6 snRNA as the endogenous enzyme. That unique selectivity even excluded as substrate U6atac RNA, the functional homolog of the minor spliceosome. Finally, RNAi knockdown experiments revealed that U6-TUTase is essential for cell proliferation. Surprisingly, large amounts of the recombinant enzyme were found to accumulate within nucleoli.

RNAi knockdown of hPrp31 leads to an accumulation of U4/U6 di-snRNPs in Cajal bodies.

Cajal bodies (CBs) are subnuclear organelles of animal and plant cells. A role of CBs in the assembly and maturation of small nuclear ribonucleoproteins (snRNP) has been proposed but is poorly understood. Here we have addressed the question where U4/U6.U5 tri-snRNP assembly occurs in the nucleus. The U4/U6.U5 tri-snRNP is a central unit of the spliceosome and must be re-formed from its components after each round of splicing. By combining RNAi and biochemical methods, we demonstrate that, after knockdown of the U4/U6-specific hPrp31 (61 K) or the U5-specific hPrp6 (102 K) protein in HeLa cells, tri-snRNP formation is inhibited and stable U5 mono-snRNPs and U4/U6 di-snRNPs containing U4/U6 proteins and the U4/U6 recycling factor p110 accumulate. Thus, hPrp31 and hPrp6 form an essential connection between the U4/U6 and U5 snRNPs in vivo. Using fluorescence microscopy, we show that, in the absence of either hPrp31 or hPrp6, U4/U6 di-snRNPs as well as p110 accumulate in Cajal bodies. In contrast, U5 snRNPs largely remain in nucleoplasmic speckles. Our data support the idea that CBs may play a role in tri-snRNP recycling.

Assembly and maturation of the U3 snoRNP in the nucleoplasm in a large dynamic multiprotein complex.

The assembly and maturation of box C/D snoRNPs, factors essential for ribosome biogenesis, occur in the nucleoplasm. To investigate this process, we have analyzed non-snoRNP factors associated with the nucleoplasmic human U3 snoRNA. We show that both the precursor and mature length nucleoplasmic U3 snoRNAs are present in larger multiprotein complexes that contain the core box C/D proteins as well as many non-snoRNP factors linked to snoRNP assembly (TIP48, TIP49, Nopp140), RNA processing (TGS1, La, LSm4, hRrp46), and subcellular localization (CRM1, PHAX). Using RNAi, we show that most of these factors are essential for box C/D snoRNA accumulation. Furthermore, we demonstrate that the core proteins undergo a restructuring event that stabilizes their binding to the snoRNA. Importantly, restructuring, which may be mediated by the putative remodeling factor TIP49, appears to be linked to nucleolar localization. We believe that the assembly complex coordinates snoRNA processing, snoRNP assembly, restructuring, and localization.

The human 18S U11/U12 snRNP contains a set of novel proteins not found in the U2-dependent spliceosome.

U11 and U12 snRNPs bind U12-type pre-mRNAs as a preformed di-snRNP complex, simultaneously recognizing the 5' splice site and branchpoint sequence. Thus, within the U12-type prespliceosome, U11/U12 components form a molecular bridge connecting both ends of the intron. We have affinity purified human 18S U11/U12 and 12S U11 snRNPs, and identified their protein components by using mass spectrometry. U11/U12 snRNPs lack all known U1 snRNP proteins but contain seven novel proteins (i.e., 65K, 59K, 48K, 35K, 31K, 25K, 20K) not found in the major spliceosome, four of which (59K, 48K, 35K, and 25K) are U11-associated. Thus, protein-protein and protein-RNA interactions contributing to 5' splice site recognition and/or intron bridging appear to differ significantly in the minor versus major prespliceosome. The majority of U11/U12 proteins are highly conserved in organisms known to contain U12-type introns. However, homologs of those associated with U11 were not detected in Drosophila melanogaster, consistent with the presence of a divergent U11 snRNP in flies. RNAi experiments revealed that several U11/U12 proteins are essential for cell viability, suggesting they play key roles in U12-type splicing. The presence of unique U11/U12 snRNP proteins in the U12-type spliceosome provides insight into potential evolutionary relationships between the major and minor spliceosome.