Pathogen Recognition and Innate Immunity

Bacteria and archaea have evolved adaptive immune defenses, termed clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems, that use short RNA to direct degradation of foreign nucleic acids. Here, we engineer the type II bacterial CRISPR system to function with custom guide RNA (gRNA) in human cells. For the endogenous AAVS1 locus, we obtained targeting rates of 10 to 25% in 293T cells, 13 to 8% in K562 cells, and 2 to 4% in induced pluripotent stem cells. We show that this process relies on CRISPR components; is sequence-specific; and, upon simultaneous introduction of multiple gRNAs, can effect multiplex editing of target loci. We also compute a genome-wide resource of ~190 K unique gRNAs targeting ~40.5% of human exons. Our results establish an RNA-guided editing tool for facile, robust, and multiplexable human genome engineering.

/pdf/rna-guided-human-genome-engineering-via-cas9-4eefu2kibr.pdf

RNA-Guided Human Genome Engineering via Cas9

The discovery of Toll-like receptors (TLRs) as components that recognize conserved structures in pathogens has greatly advanced understanding of how the body senses pathogen invasion, triggers innate immune responses and primes antigen-specific adaptive immunity. Although TLRs are critical for host defense, it has become apparent that loss of negative regulation of TLR signaling, as well as recognition of self molecules by TLRs, are strongly associated with the pathogenesis of inflammatory and autoimmune diseases. Furthermore, it is now clear that the interaction between TLRs and recently identified cytosolic innate immune sensors is crucial for mounting effective immune responses. Here we describe the recent advances that have been made by research into the role of TLR biology in host defense and disease.

/pdf/the-role-of-pattern-recognition-receptors-in-innate-immunity-39y77tlxza.pdf

The role of pattern-recognition receptors in innate immunity: update on Toll-like receptors

Pattern Recognition Receptors and Inflammation

The transdifferentiation of epithelial cells into motile mesenchymal cells, a process known as epithelial-mesenchymal transition (EMT), is integral in development, wound healing and stem cell behaviour, and contributes pathologically to fibrosis and cancer progression. This switch in cell differentiation and behaviour is mediated by key transcription factors, including SNAIL, zinc-finger E-box-binding (ZEB) and basic helix-loop-helix transcription factors, the functions of which are finely regulated at the transcriptional, translational and post-translational levels. The reprogramming of gene expression during EMT, as well as non-transcriptional changes, are initiated and controlled by signalling pathways that respond to extracellular cues. Among these, transforming growth factor-β (TGFβ) family signalling has a predominant role; however, the convergence of signalling pathways is essential for EMT.

Molecular mechanisms of epithelial–mesenchymal transition

Antisense oligonucleotides (ODNs) are powerful tools with which to determine the consequences of the reduced expression of a selected target gene, and they may have important therapeutic applications. Methods for predicting optimum antisense sites are not always effective because various factors, such as RNA-binding proteins, influence the secondary and tertiary structures of RNAs in vivo. To overcome this obstacle, we have attempted to engineer an antisense system that can unravel secondary and tertiary RNA structures. To create such an antisense system, we connected the constitutive transport element (CTE), an RNA motif that has the ability to interact with intracellular RNA helicases, to an antisense sequence so that helicase-binding hybrid antisense ODN would be produced in cells. We postulated that this modification would enhance antisense activity in vivo, with more frequent hybridization of the antisense ODN with its targeting site. Western blotting analysis demonstrated that a hybrid antisense ODN targeted to the bcl-2 gene suppressed the expression of this gene more effectively than did the antisense ODN alone. Our results suggest that the effects of antisense ODNs can be enhanced when their actions are combined with those of RNA helicases.

Stimulatory effect of an indirectly attached RNA helicase-recruiting sequence on the suppression of gene expression by antisense oligonucleotides.

Unspliced XBP1 contributes to cholesterol biosynthesis and tumorigenesis by stabilizing SREBP2 in hepatocellular carcinoma

A method of inhibiting the replication ability of a hepatitis C virus (HCV) provided. An oligoribonucleotide or a peptide nucleic acid which sequence-specifically binds to the HCV-RNA, and a therapeutic agent for bepatitis C which contains any of these components as an active ingredient are provided.

Oligoribonucleotide or peptide nucleic acid which inhibits action of hepatitis C virus

A double-stranded RNA for interference comprising an antisense RNA being complementary to a sequence in the neighborhood region of a target gene or a sequence in any of regions in mRNA of the target gene and a sense RNA homologous with the above sequence which is hybridizable with the above-described antisense RNA in a cell, wherein the above-described antisense RNA and the above-described sense RNA form double-strands and the double-stranded moiety has a length consisting of 50 or more bases. Use of this long double-stranded RNA for interference makes it possible to highly efficiently induce RNAi with a lowered cytotoxicity without resort to attempts to determine the part to be used as a sequence complementary to the antisense RNA. By introducing a base substitution or bulge into such a long double-stranded RNA for interference, the effects of the RNAi can be further enhanced and interferon response can be lowered.

Long double-stranded rna for interference with lowered interferon response

A multiple shRNA expression vector for use in the inhibition of the expression of a target gene, which carries multiple shRNA-producing units each of which can produce shRNA. In the vector, each of the shRNA-producing units comprises DNA encoding shRNA ligated downstream to a promoter, the DNA encoding the shRNA is composed of antisense DNA encoding antisense RNA which has a sequence complementary to mRNA of the target gene and sense DNA encoding sense RNA having a sequence hybridizable with the antisense DNA, and both of the antisense DNA and the sense DNA are ligated to each other through linker DNA.

Makoto Miyagishi

Papers

Stimulatory effect of an indirectly attached RNA helicase-recruiting sequence on the suppression of gene expression by antisense oligonucleotides.

Unspliced XBP1 contributes to cholesterol biosynthesis and tumorigenesis by stabilizing SREBP2 in hepatocellular carcinoma

Oligoribonucleotide or peptide nucleic acid which inhibits action of hepatitis C virus

Long double-stranded rna for interference with lowered interferon response

MULTIPLE shRNA EXPRESSION VECTOR