Circular RNAs composed of exonic sequence have been described in a small number of genes. Thought to result from splicing errors, circular RNA species possess no known function. To delineate the universe of endogenous circular RNAs, we performed high-throughput sequencing (RNA-seq) of libraries prepared from ribosome-depleted RNA with or without digestion with the RNA exonuclease, RNase R. We identified >25,000 distinct RNA species in human fibroblasts that contained non-colinear exons (a "backsplice") and were reproducibly enriched by exonuclease degradation of linear RNA. These RNAs were validated as circular RNA (ecircRNA), rather than linear RNA, and were more stable than associated linear mRNAs in vivo. In some cases, the abundance of circular molecules exceeded that of associated linear mRNA by >10-fold. By conservative estimate, we identified ecircRNAs from 14.4% of actively transcribed genes in human fibroblasts. Application of this method to murine testis RNA identified 69 ecircRNAs in precisely orthologous locations to human circular RNAs. Of note, paralogous kinases HIPK2 and HIPK3 produce abundant ecircRNA from their second exon in both humans and mice. Though HIPK3 circular RNAs contain an AUG translation start, it and other ecircRNAs were not bound to ribosomes. Circular RNAs could be degraded by siRNAs and, therefore, may act as competing endogenous RNAs. Bioinformatic analysis revealed shared features of circularized exons, including long bordering introns that contained complementary ALU repeats. These data show that ecircRNAs are abundant, stable, conserved and nonrandom products of RNA splicing that could be involved in control of gene expression.

/pdf/circular-rnas-are-abundant-conserved-and-associated-with-alu-3965fj7kwv.pdf

Circular RNAs are abundant, conserved, and associated with ALU repeats

Interleukin-10 (IL-10), a cytokine with anti-inflammatory properties, has a central role in infection by limiting the immune response to pathogens and thereby preventing damage to the host. Recently, an increasing interest in how IL10 expression is regulated in different immune cells has revealed some of the molecular mechanisms involved at the levels of signal transduction, epigenetics, transcription factor binding and gene activation. Understanding the specific molecular events that regulate the production of IL-10 will help to answer the remaining questions that are important for the design of new strategies of immune intervention.

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The regulation of IL-10 production by immune cells

Rapid improvements in sequencing and array-based platforms are resulting in a flood of diverse genome-wide data, including data from exome and whole-genome sequencing, epigenetic surveys, expression profiling of coding and noncoding RNAs, single nucleotide polymorphism (SNP) and copy number profiling, and functional assays. Analysis of these large, diverse data sets holds the promise of a more comprehensive understanding of the genome and its relation to human disease. Experienced and knowledgeable human review is an essential component of this process, complementing computational approaches. This calls for efficient and intuitive visualization tools able to scale to very large data sets and to flexibly integrate multiple data types, including clinical data. However, the sheer volume and scope of data pose a significant challenge to the development of such tools.

Integrative Genomics Viewer

Circular RNA transcripts were first identified in the early 1990s but knowledge of these species has remained limited, as their study through traditional methods of RNA analysis has been difficult Now, novel bioinformatic approaches coupled with biochemical enrichment strategies and deep sequencing have allowed comprehensive studies of circular RNA species Recent studies have revealed thousands of endogenous circular RNAs in mammalian cells, some of which are highly abundant and evolutionarily conserved Evidence is emerging that some circRNAs might regulate microRNA (miRNA) function, and roles in transcriptional control have also been suggested Therefore, study of this class of noncoding RNAs has potential implications for therapeutic and research applications We believe the key future challenge for the field will be to understand the regulation and function of these unusual molecules

Detecting and characterizing circular RNAs

Circular Intronic Long Noncoding RNAs

Toll-like receptors (TLRs) are a family of proteins playing important roles in host defense. Mice defective of functional TLR4 are hyporesponsive to LPS, suggesting that TLR4 is essential for LPS signaling. Here we report the cloning of an alternatively spliced mouse TLR4 (mTLR4) mRNA. The additional exon exists between the second and third exon of the reported mTLR4 gene and contains an in-frame stop codon. The alternatively spliced mRNA encodes 86 aa of the reported mTLR4 and an additional 36 aa. This alternatively spliced mTLR4 mRNA expressed a partially secretary 20-kDa protein, which we named soluble mTLR4 (smTLR4). In a mouse macrophage cell line, the exogenously expressed smTLR4 significantly inhibited LPS-mediated TNF-alpha production and NF-kappaB activation. Additionally, in mouse macrophages, LPS increased the mRNA for smTLR4. Taken together, our results indicate that smTLR4 may function as a feedback mechanism to inhibit the excessive LPS responses in mouse macrophages.

Cutting edge: Naturally occurring soluble form of mouse toll-like receptor 4 inhibits lipopolysaccharide signaling

Yeast carries a strictly conserved branch point sequence (BPS) of UACUAAC, whereas the human BPS is degenerative and is less well characterized. The human consensus BPS has never been extensively explored in vitro to date. Here, we sequenced 367 clones of lariat RT-PCR products arising from 52 introns of 20 human housekeeping genes. Among the 367 clones, a misincorporated nucleotide at the branch point was observed in 181 clones, for which we can precisely pinpoint the branch point. The branch points were comprised of 92.3% A, 3.3% C, 1.7% G and 2.8% U. Our analysis revealed that the human consensus BPS is simply yUnAy, where the underlined is the branch point at position zero and the lowercase pyrimidines (‘y’) are not as well conserved as the uppercase U and A. We found that the branch points are located 21–34 nucleotides upstream of the 3′ end of an intron in 83% clones. We also found that the polypyrimidine tract spans 4–24 nucleotides downstream of the branch point. Our analysis demonstrates that the human BPSs are more degenerative than we have expected and that the human BPSs are likely to be recognized in combination with the polypyrimidine tract and/or the other splicing cis-elements.

/pdf/human-branch-point-consensus-sequence-is-yunay-2mqpa1s0e1.pdf

Human branch point consensus sequence is yUnAy

FUS is an RNA-binding protein that regulates transcription, alternative splicing, and mRNA transport. Aberrations of FUS are causally associated with familial and sporadic ALS/FTLD. We analyzed FUS-mediated transcriptions and alternative splicing events in mouse primary cortical neurons using exon arrays. We also characterized FUS-binding RNA sites in the mouse cerebrum with HITS-CLIP. We found that FUS-binding sites tend to form stable secondary structures. Analysis of position-dependence of FUS-binding sites disclosed scattered binding of FUS to and around the alternatively spliced exons including those associated with neurodegeneration such as Mapt, Camk2a, and Fmr1. We also found that FUS is often bound to the antisense RNA strand at the promoter regions. Global analysis of these FUS-tags and the expression profiles disclosed that binding of FUS to the promoter antisense strand downregulates transcriptions of the coding strand. Our analysis revealed that FUS regulates alternative splicing events and transcriptions in a position-dependent manner.

/pdf/position-dependent-fus-rna-interactions-regulate-alternative-4p1pg4mjgj.pdf

Position-dependent FUS-RNA interactions regulate alternative splicing events and transcriptions

Molecular hydrogen is protective against 6-hydroxydopamine-induced nigrostriatal degeneration in a rat model of Parkinson's disease.

Mast cells secrete multiple cytokines and play an important role in allergic inflammation. Although it is widely accepted that bacteria infection occasionally worsens allergic airway inflammation, the mechanism has not been defined. In this study, we show that LPS induced Th2-associated cytokine production such as IL-5, IL-10, and IL-13 from mast cells and also synergistically enhanced production of these cytokines induced by IgE cross-linking. LPS-mediated Th2-type cytokine production was abolished in mouse bone marrow-derived mast cells derived from C3H/HeJ mice, suggesting that Toll-like receptor 4 is essential for the cytokine production. Furthermore, we found that mitogen-activated protein kinases including extracellular signal-regulated kinase 1/2, c-Jun N-terminal kinase, and p38 kinase were activated by LPS stimulation in bone marrow-derived mast cells. Inhibition of extracellular signal-regulated kinase activation has little effect on LPS-mediated cytokine production. In contrast, inhibition of c-Jun N-terminal kinase activation significantly suppressed both IL-10 and IL-13 expression at both mRNA and protein levels. Interestingly, although inhibition of p38 did not down-regulate the mRNA induction, it moderately decreased all three cytokine productions by LPS. These results indicate that LPS-mediated production of IL-5, IL-10, and IL-13 was distinctly regulated by mitogen-activated protein kinases. Our findings may indicate a clue to understanding the mechanisms of how bacteria infection worsens the clinical features of asthma.

/pdf/th2-cytokine-production-from-mast-cells-is-directly-induced-53ziscj07q.pdf

Th2 cytokine production from mast cells is directly induced by lipopolysaccharide and distinctly regulated by c-Jun N-terminal kinase and p38 pathways.

Akio Masuda

Papers

Cutting edge: Naturally occurring soluble form of mouse toll-like receptor 4 inhibits lipopolysaccharide signaling

Human branch point consensus sequence is yUnAy

Position-dependent FUS-RNA interactions regulate alternative splicing events and transcriptions

Molecular hydrogen is protective against 6-hydroxydopamine-induced nigrostriatal degeneration in a rat model of Parkinson's disease.

Th2 cytokine production from mast cells is directly induced by lipopolysaccharide and distinctly regulated by c-Jun N-terminal kinase and p38 pathways.