Molecular Mechanisms of Alternative Splicing Regulation: An Investigation of the Spliceosome Repressed by Hnrnp L on Cd45 Exon 4

Alternative splicing is a key step in gene regulation and involves the differential selection of splice sites to generate different pre-mRNA transcripts. It has been shown that 90-95% of pre-mRNAs are alternatively spliced in human cells. Pre-mRNA splicing is catalyzed the spliceosome, which consists mainly of the U1, U2, U4, U5 and U6 snRNP, and about a hundred of non-snRNP proteins. Splicing regulators that bind to enhancer or silencer elements on the pre-mRNA can alter assembly of these spliceosome components. Understanding how splicing regulators control spliceosome assembly will bring insights to the prediction of splice site choices. In our lab, we used CD45 as a model gene for studying alternative splicing and spliceosome assembly. The exonic silencer sequence (ESS1) within CD45 exon 4 is bound by hnRNP L to induce its skipping. HnRNP L represses spliceosome assembly at a step after the binding of U1 and U2 snRNP on either side of the exon. My goal is to understand how hnRNP L perturbs U1 snRNP binding at the 5\u27 splice site (5\u27 ss) to cause the skipping of CD45 exon 4. Using psoralen- and UV-crosslinking analysis, U1 snRNP and other protein components that associate with the sequences around the 5\u27 ss within the hnRNP L-repressed spliceosome complexes were compared with the control complexes. These studies revealed that hnRNP L recruits hnRNP A1 to the 3\u27 end of exon 4 to induce an extended pairing interaction between the U1 snRNA and the 5\u27 ss. Splicing assays in vitro and in cells further demonstrated that hnRNP A1 and the U1 snRNA binding at the 3\u27end of exon 4 are required for hnRNP L-mediated skipping of exon 4. Further analysis of other exons repressed by hnRNP L or A1 suggests the potential for the extended U1 pairing interactions with these exons. These data imply that induction of U1 interactions with the exonic region nearby a 5\u27 ss could be a widespread mechanism in inducing the skipping of exon. To further determine how the extended U1 binding affects the subsequent spliceosome assembly steps, hnRNP L-repressed spliceosome complexes were purified. This analysis revealed that association of U6 snRNP with the 5\u27ss, and recruitment of NTC components, are blocked in the hnRNP L-repressed complexes. Moreover, enhancing binding of U6 to the 5\u27ss overcomes the effect of the extended U1 interaction, thereby increasing the splicing of the hnRNP L-repressed substrate. These results provide the first example showing that the U1/U6 switch, a structural rearrangement during the catalytic activation of the spliceosome, is a naturally occurring point for regulating alternative splicing. This study also suggests that splicing regulators that alter U1 binding, or spliceosome components that associate with the 5\u27 ss after U1 binding, are important factors for determining the usage of the 5\u27ss

Increased Hematopoietic Extracellular RNAs and Vesicles in the Lung during Allergic Airway Responses.

Extracellular RNAs (exRNAs) can be released by numerous cell types in vitro, are often protected within vesicles, and can modify recipient cell function. To determine how the composition and cellular sources of exRNAs and the extracellular vesicles (EVs) that carry them change in vivo during tissue inflammation, we analyzed bronchoalveolar lavage fluid (BALF) from mice before and after lung allergen challenge. In the lung, extracellular microRNAs (ex-miRNAs) had a composition that was highly correlated with airway-lining epithelium. Using cell type-specific membrane tagging and single vesicle flow, we also found that 80% of detected vesicles were of epithelial origin. After the induction of allergic airway inflammation, miRNAs selectively expressed by immune cells, including miR-223 and miR-142a, increased and hematopoietic-cell-derived EVs also increased >2-fold. These data demonstrate that infiltrating immune cells release ex-miRNAs and EVs in inflamed tissues to alter the local extracellular environment

First Ground Observations of OI5577 Green Line Emission over the Taiwan Area

Worldwide ground observations of upper atmospheric airglow with particular emphasis on the OI 557.7 and 630 nm emissions have been conducted since 1960s. This study reports the first ground observations of OI 557.7 nm green line emission over the Taiwan area. For comparison, the background continuum at 530 nm was also measured by the same system. The experiments were conducted during the period of Aug - Dec, 2004 at various locations in Taiwan using a self-developed photometer instrument. Daily height integrated intensity of the night-time green line emission may vary in the range of 80 - 210 Rayleighs and twilight enhancement is also identified. The observational results may serve as a useful reference for follow-up sounding rocket measurements of OI 557.7 nm airglow emission over the Taiwan area

Association of combination antiretroviral therapy with risk of neurological diseases in patients with HIV/AIDS in Taiwan: a nested case-control study

Heterogeneous neurocognitive impairment remains an important issue, even in the era of combination antiretroviral therapy (cART), with an incidence ranging from 15% to 65%. Although ART drugs with higher penetration scores to the central nervous system (CNS) show better HIV replication control in the CNS, the association between CNS penetration effectiveness (CPE) scores and neurocognitive impairment remains inconclusive. To explore whether ART exposure is associated with the risk of neurological diseases among patients with HIV/AIDS, this study in Taiwan involved 2,571 patients with neurological diseases and 10,284 matched, randomly selected patients without neurological diseases between 2010 and 2017. A conditional logistic regression model was used in this study. The parameters for ART exposure included ART usage, timing of exposure, cumulative defined daily dose (DDD), adherence, and cumulative CPE score. Incident cases of neurological diseases, including CNS infections, cognitive disorders, vasculopathy, and peripheral neuropathy, were obtained from the National Health Insurance Research Database in Taiwan. Odds ratios (ORs) for the risk of neurological diseases were conducted using a multivariate conditional logistic regression model. Patients with a history of past exposure (OR: 1.68, 95% confidence interval [CI]:1.22–2.32), low cumulative DDDs (< 2,500) (OR: 1.28, 95% CI: 1.15–1.42), low adherence (0 < adherence (ADH) ≤ 0.8) (OR: 1.46, 95% CI: 1.30–1.64), or high cumulative CPE scores (>14) (OR: 1.34, 95% CI: 1.14–1.57) had a high risk of neurological diseases. When stratified by classes of ART drugs, patients with low cumulative DDDs or low adherence had a high risk of neurological diseases, including NRTIs, PIs, NNRTIs, INSTIs, and multi-drug tablets. Subgroup analyses also suggested that patients with low cumulative DDDs or low adherence had a high risk of neurological diseases when they had high cumulative CPE scores. Patients with high cumulative DDDs or medication adherence were protected against neurological diseases only when they had low cumulative CPE scores (≤ 14). Patients may be at risk for neurological diseases when they have low cumulative DDDs, low adherence, or usage with high cumulative CPE scores. Continuous usage and low cumulative CPE scores of ART drugs may benefit neurocognitive health in patients with HIV/AIDS

Large expert-curated database for benchmarking document similarity detection in biomedical literature search

Document recommendation systems for locating relevant literature have mostly relied on methods developed a decade ago. This is largely due to the lack of a large offline gold-standard benchmark of relevant documents that cover a variety of research fields such that newly developed literature search techniques can be compared, improved and translated into practice. To overcome this bottleneck, we have established the RElevant LIterature SearcH consortium consisting of more than 1500 scientists from 84 countries, who have collectively annotated the relevance of over 180 000 PubMed-listed articles with regard to their respective seed (input) article/s. The majority of annotations were contributed by highly experienced, original authors of the seed articles. The collected data cover 76% of all unique PubMed Medical Subject Headings descriptors. No systematic biases were observed across different experience levels, research fields or time spent on annotations. More importantly, annotations of the same document pairs contributed by different scientists were highly concordant. We further show that the three representative baseline methods used to generate recommended articles for evaluation (Okapi Best Matching 25, Term Frequency-Inverse Document Frequency and PubMed Related Articles) had similar overall performances. Additionally, we found that these methods each tend to produce distinct collections of recommended articles, suggesting that a hybrid method may be required to completely capture all relevant articles. The established database server located at https://relishdb.ict.griffith.edu.au is freely available for the downloading of annotation data and the blind testing of new methods. We expect that this benchmark will be useful for stimulating the development of new powerful techniques for title and title/abstract-based search engines for relevant articles in biomedical research.Peer reviewe

Selective Export into Extracellular Vesicles and Function of tRNA Fragments during T Cell Activation

Summary: The discovery of microRNA (miRNA) sorting into extracellular vesicles (EVs) revealed a novel mode of intercellular communication and uncovered a link between cellular endomembrane compartments and small RNAs in EV-secreting cells. Using a two-step ultracentrifugation procedure to isolate EVs released by T cells, we found that 45% of tRNA fragments (tRFs), but fewer than 1% of miRNAs, were significantly enriched in EVs compared with the corresponding cellular RNA. T cell activation induced the EV-mediated release of a specific set of tRFs derived from the 5′ end and 3′-internal region of tRNAs without variable loops. Inhibition of EV biogenesis pathways specifically led to the accumulation of these activation-induced EV-enriched tRFs within multivesicular bodies (MVBs). Introducing antisense oligonucleotides to inhibit these tRFs enhanced T cell activation. Taken together, these results demonstrate that T cells selectively release tRFs into EVs via MVBs and suggest that this process may remove tRFs that repress immune activation. : Chiou et al. show that T cells release extracellular vesicles that carry RNA cargo enriched in tRNA fragments. Immune activating signals enhance multivesicular body formation and the secretion of vesicles containing specific tRNA fragments. Within cells, these tRNA fragments inhibit T cell activation and cytokine production. Keywords: tRNA fragment, tsRNA, extracellular vesicle, exosome, T lymphocyt

Cellular RNA Binding Proteins NS1-BP and hnRNP K Regulate Influenza A Virus RNA Splicing

<div><p>Influenza A virus is a major human pathogen with a genome comprised of eight single-strand, negative-sense, RNA segments. Two viral RNA segments, NS1 and M, undergo alternative splicing and yield several proteins including NS1, NS2, M1 and M2 proteins. However, the mechanisms or players involved in splicing of these viral RNA segments have not been fully studied. Here, by investigating the interacting partners and function of the cellular protein NS1-binding protein (NS1-BP), we revealed novel players in the splicing of the M1 segment. Using a proteomics approach, we identified a complex of RNA binding proteins containing NS1-BP and heterogeneous nuclear ribonucleoproteins (hnRNPs), among which are hnRNPs involved in host pre-mRNA splicing. We found that low levels of NS1-BP specifically impaired proper alternative splicing of the viral M1 mRNA segment to yield the M2 mRNA without affecting splicing of mRNA₃, M4, or the NS mRNA segments. Further biochemical analysis by formaldehyde and UV cross-linking demonstrated that NS1-BP did not interact directly with viral M1 mRNA but its interacting partners, hnRNPs A1, K, L, and M, directly bound M1 mRNA. Among these hnRNPs, we identified hnRNP K as a major mediator of M1 mRNA splicing. The M1 mRNA segment generates the matrix protein M1 and the M2 ion channel, which are essential proteins involved in viral trafficking, release into the cytoplasm, and budding. Thus, reduction of NS1-BP and/or hnRNP K levels altered M2/M1 mRNA and protein ratios, decreasing M2 levels and inhibiting virus replication. Thus, NS1-BP-hnRNPK complex is a key mediator of influenza A virus gene expression.</p></div