A mutation in the low-complexity domain of splicing factor hnRNPA1 linked to amyotrophic lateral sclerosis disrupts distinct neuronal RNA splicing networks.

Amyotrophic lateral sclerosis (ALS) is a debilitating neurodegenerative disease characterized by loss of motor neurons. Human genetic studies have linked mutations in RNA-binding proteins as causative for this disease. The hnRNPA1 protein, a known pre-mRNA splicing factor, is mutated in some ALS patients. Here, two human cell models were generated to investigate how a mutation in the C-terminal low-complexity domain (LCD) of hnRNPA1 can cause splicing changes of thousands of transcripts that collectively are linked to the DNA damage response, cilium organization, and translation. We show that the hnRNPA1 D262V mutant protein binds to new binding sites on differentially spliced transcripts from genes that are linked to ALS. We demonstrate that this ALS-linked hnRNPA1 mutation alters normal RNA-dependent protein-protein interactions. Furthermore, cells expressing this hnRNPA1 mutant exhibit a cell aggregation phenotype, markedly reduced growth rates, changes in stress granule kinetics, and aberrant growth of neuronal processes. This study provides insight into how a single amino acid mutation in a splicing factor can alter RNA splicing networks of genes linked to ALS.

Coordinate regulation of alternative pre-mRNA splicing events by the human RNA chaperone proteins hnRNPA1 and DDX5.

Alternative premessenger RNA (pre-mRNA) splicing is a post-transcriptional mechanism for controlling gene expression. Splicing patterns are determined by both RNA-binding proteins and nuclear pre-mRNA structure. Here, we analyzed pre-mRNA splicing patterns, RNA-binding sites, and RNA structures near these binding sites coordinately controlled by two splicing factors: the heterogeneous nuclear ribonucleoprotein hnRNPA1 and the RNA helicase DDX5. We identified thousands of alternative pre-mRNA splicing events controlled by these factors by RNA sequencing (RNA-seq) following RNAi. Enhanced cross-linking and immunoprecipitation (eCLIP) on nuclear extracts was used to identify protein-RNA-binding sites for both proteins in the nuclear transcriptome. We found a significant overlap between hnRNPA1 and DDX5 splicing targets and that they share many closely linked binding sites as determined by eCLIP analysis. In vivo SHAPE (selective 2'-hydroxyl acylation analyzed by primer extension) chemical RNA structure probing data were used to model RNA structures near several exons controlled and bound by both proteins. Both sequence motifs and in vivo UV cross-linking sites for hnRNPA1 and DDX5 were used to map binding sites in their RNA targets, and often these sites flanked regions of higher chemical reactivity, suggesting an organized nature of nuclear pre-mRNPs. This work provides a first glimpse into the possible RNA structures surrounding pre-mRNA splicing factor-binding sites.

Patterns in the Reporting of Aggressive Histologic Subtypes in Papillary Thyroid Cancer.

INTRODUCTION: The tall cell, columnar, and diffuse sclerosing subtypes are aggressive histologic subtypes of papillary thyroid cancer (PTC) with increasing incidence, yet there is a wide variation in reporting. We aimed to identify and compare factors associated with the reporting of these aggressive subtypes (aPTC) to classic PTC (cPTC) and secondarily identify differences in outcomes. METHODS: The National Cancer Database was utilized to identify cPTC and aPTC from 2004 to 2017. Patient and facility demographics and clinicopathologic variables were analyzed. Independent predictors of aPTC reporting were identified and a survival analysis was performed. RESULTS: The majority of aPTC (67%) were reported by academic facilities. Compared to academic facilities, all other facility types were 1.4-2.0 times less likely to report aPTC (P < 0.05). Regional variation in reporting was noted, with more cases reported in the Middle Atlantic, despite there being more total facilities in the South Atlantic and East North Central regions. Compared to the Middle Atlantic, all other regions were 1.4-5 times less likely to report aPTC (P < 0.001). Patient characteristics including race and income were not associated with aPTC reporting. Compared to cPTC, aPTC had higher rates of aggressive features and worse 5-y overall survival (90.5% versus 94.5%, log rank P < 0.001). CONCLUSIONS: Aggressive subtypes of PTC are associated with worse outcomes. Academic and other facilities in the Middle Atlantic were more likely to report aPTC. This suggests the need for further evaluation of environmental or geographic factors versus a need for increased awareness and more accurate diagnosis of these subtypes.

PD-L1 expression and infiltration by CD4+ and FoxP3+ T cells are increased in Xp11 translocation renal cell carcinoma and indicate poor prognosis.

AIMS: Interaction between programmed death-1 ligand (PD-L1) and its receptor programmed death 1 (PD-1) on T cells inactivates antitumour immune responses. PD-L1 expression has been associated with poor prognosis in renal cell carcinoma (RCC) and predicts adverse outcome. This study was designed to evaluate the impact of PD-L1 expression and the immune microenvironment on the clinical outcome in Xp11 translocation renal cell carcinoma (TRCC) and, therefore, their potential relevance as prognostic biomarkers. METHODS AND RESULTS: The present retrospective analysis investigated expression of PD-L1 and immune cells CD8, CD4, CD3, forkhead box protein 3 (FoxP3) and PD-1 in TRCC compared to other types of RCC. FFPE specimens were collected between 2011 and 2017 from 311 patients who underwent nephrectomy at our institution for RCC. Specimens were immunostained for PD-L1, CD8, CD4, CD3, FoxP3 and PD-1, and an outcome analysis was conducted. PD-L1 expression rate was highest in TRCC (68%, 16 of 25), followed by mucinous tubular and spindle cell RCC and collecting duct carcinoma (33%, one of three), papillary RCC (27%, seven of 26), clear cell RCC (16%, 29 of 233), chromophobe RCC (11%, two of 18) and multilocular cystic RCC (0%, none of three). In TRCC, PD-L1 expression was associated with poor recurrence-free survival (RFS) (P = 0.041). The CD4high and FoxP3high groups showed a significantly shorter RFS (P = 0.05 and P = 0.031, respectively) compared to CD4low and FOXPlow groups. CONCLUSION: PD-L1 expression was higher in TRCC than in other types of RCC. High PD-L1 tumour cell expression and tumour infiltration by CD4+ and FoxP3+ immune cells were associated with poor RFS in TRCC.

Efficient CRISPR-rAAV engineering of endogenous genes to study protein function by allele-specific RNAi.

Gene knockout strategies, RNAi and rescue experiments are all employed to study mammalian gene function. However, the disadvantages of these approaches include: loss of function adaptation, reduced viability and gene overexpression that rarely matches endogenous levels. Here, we developed an endogenous gene knockdown/rescue strategy that combines RNAi selectivity with a highly efficient CRISPR directed recombinant Adeno-Associated Virus (rAAV) mediated gene targeting approach to introduce allele-specific mutations plus an allele-selective siRNA Sensitive (siSN) site that allows for studying gene mutations while maintaining endogenous expression and regulation of the gene of interest. CRISPR/Cas9 plus rAAV targeted gene-replacement and introduction of allele-specific RNAi sensitivity mutations in the CDK2 and CDK1 genes resulted in a >85% site-specific recombination of Neo-resistant clones versus ∼8% for rAAV alone. RNAi knockdown of wild type (WT) Cdk2 with siWT in heterozygotic knockin cells resulted in the mutant Cdk2 phenotype cell cycle arrest, whereas allele specific knockdown of mutant CDK2 with siSN resulted in a wild type phenotype. Together, these observations demonstrate the ability of CRISPR plus rAAV to efficiently recombine a genomic locus and tag it with a selective siRNA sequence that allows for allele-selective phenotypic assays of the gene of interest while it remains expressed and regulated under endogenous control mechanisms.

Aberrant herpesvirus-induced polyadenylation correlates with cellular messenger RNA destruction.

Regulation of messenger RNA (mRNA) stability plays critical roles in controlling gene expression, ensuring transcript fidelity, and allowing cells to respond to environmental cues. Unregulated enhancement of mRNA turnover could therefore dampen cellular responses to such signals. Indeed, several herpesviruses instigate widespread destruction of cellular mRNAs to block host gene expression and evade immune detection. Kaposi's sarcoma-associated herpesvirus (KSHV) promotes this phenotype via the activity of its viral SOX protein, although the mechanism of SOX-induced mRNA turnover has remained unknown, given its apparent lack of intrinsic ribonuclease activity. Here, we report that KSHV SOX stimulates cellular transcriptome turnover via a unique mechanism involving aberrant polyadenylation. Transcripts in SOX-expressing cells exhibit extended poly(A) polymerase II-generated poly(A) tails and polyadenylation-linked mRNA turnover. SOX-induced polyadenylation changes correlate with its RNA turnover function, and inhibition of poly(A) tail formation blocks SOX activity. Both nuclear and cytoplasmic poly(A) binding proteins are critical cellular cofactors for SOX function, the latter of which undergoes striking nuclear relocalization by SOX. SOX-induced mRNA turnover therefore represents both a novel mechanism of host shutoff as well as a new model system to probe the regulation of poly(A) tail-stimulated mRNA turnover in mammalian cells.

Metastatic pancreatic neuroendocrine tumors feature elevated T cell infiltration.

Pancreatic neuroendocrine tumors (PNETs) are malignancies arising from the islets of Langerhans. Therapeutic options are limited for the over 50% of patients who present with metastatic disease. We aimed to identify mechanisms to remodel the PNET tumor microenvironment (TME) to ultimately enhance susceptibility to immunotherapy. The TMEs of localized and metastatic PNETs were investigated using an approach that combines RNA-Seq, cancer and T cell profiling, and pharmacologic perturbations. RNA-Seq analysis indicated that the primary tumors of metastatic PNETs showed significant activation of inflammatory and immune-related pathways. We determined that metastatic PNETs featured increased numbers of tumor-infiltrating T cells compared with localized tumors. T cells isolated from both localized and metastatic PNETs showed evidence of recruitment and antigen-dependent activation, suggestive of an immune-permissive microenvironment. A computational analysis suggested that vorinostat, a histone deacetylase inhibitor, may perturb the transcriptomic signature of metastatic PNETs. Treatment of PNET cell lines with vorinostat increased chemokine CCR5 expression by NF-κB activation. Vorinostat treatment of patient-derived metastatic PNET tissues augmented recruitment of autologous T cells, and this augmentation was substantiated in a mouse model of PNET. Pharmacologic induction of chemokine expression may represent a promising approach for enhancing the immunogenicity of metastatic PNET TMEs.

A Cdk4/6-dependent phosphorylation gradient regulates the early to late G1 phase transition.

During early G1 phase, Rb is exclusively mono-phosphorylated by cyclin D:Cdk4/6, generating 14 different isoforms with specific binding patterns to E2Fs and other cellular protein targets. While mono-phosphorylated Rb is dispensable for early G1 phase progression, interfering with cyclin D:Cdk4/6 kinase activity prevents G1 phase progression, questioning the role of cyclin D:Cdk4/6 in Rb inactivation. To dissect the molecular functions of cyclin D:Cdk4/6 during cell cycle entry, we generated a single cell reporter for Cdk2 activation, RB inactivation and cell cycle entry by CRISPR/Cas9 tagging endogenous p27 with mCherry. Through single cell tracing of Cdk4i cells, we identified a time-sensitive early G1 phase specific Cdk4/6-dependent phosphorylation gradient that regulates cell cycle entry timing and resides between serum-sensing and cyclin E:Cdk2 activation. To reveal the substrate identity of the Cdk4/6 phosphorylation gradient, we performed whole proteomic and phospho-proteomic mass spectrometry, and identified 147 proteins and 82 phospho-peptides that significantly changed due to Cdk4 inhibition in early G1 phase. In summary, we identified novel (non-Rb) cyclin D:Cdk4/6 substrates that connects early G1 phase functions with cyclin E:Cdk2 activation and Rb inactivation by hyper-phosphorylation.

The phosphorylation state of both hERG and KvLQT1 mediates protein-protein interactions between these complementary cardiac potassium channel alpha subunits.

KvLQT1 and hERG are the α-subunits of the voltage-gated K+ channels which carry the cardiac repolarizing currents IKs and IKr, respectively. These currents function in vivo with some redundancy to maintain appropriate action potential durations (APDs) in cardiomyocytes. As such, protein-protein interactions between hERG and KvLQT1 may be important in normal cardiac electrophysiology, as well as in arrhythmia and sudden cardiac death. Previous phenomenological observations of functional, mutual downregulation between these complementary repolarizing currents in transgenic rabbit models and human cell culture motivate our investigations into protein-protein interactions between hERG and KvLQT1. Previous data suggest that a dynamic, physical interaction between hERG and KvLQT1 modulates the respective currents. However, the mechanism by which hERG-KvLQT1 interactions are regulated is still poorly understood. Phosphorylation is proposed to play a role since modifying the phosphorylation state of each protein has been shown to alter channel kinetics, and both hERG and KvLQT1 are targets of the Ser/Thr protein kinase PKA, activated by elevated intracellular cAMP. In this work, quantitative apFRET analyses of phosphonull and phosphomimetic hERG and KvLQT1 mutants indicate that unphosphorylated hERG does not interact with KvLQT1, suggesting that hERG phosphorylation is important for wild-type proteins to interact. For proteins already potentially interacting, phosphorylation of KvLQT1 appears to be the driving factor abrogating hERG-KvLQT1 interaction. This work increases our knowledge about hERG-KvLQT1 interactions, which may contribute to the efforts to elucidate mechanisms that underlie many types of arrhythmias, and also further characterizes novel protein-protein interactions between two distinct potassium channel families.

Host shutoff is a conserved phenotype of gammaherpesvirus infection and is orchestrated exclusively from the cytoplasm.

Lytic infection with the two human gammaherpesviruses, Kaposi's sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV), leads to significant depletion of the cellular transcriptome. This host shutoff phenotype is driven by the conserved herpesviral alkaline exonuclease, termed SOX in KSHV and BGLF5 in EBV, which in gammaherpesviruses has evolved the genetically separable ability to target cellular mRNA. We now show that host shutoff is also a prominent consequence of murine gammaherpesvirus 68 (MHV68) infection, which is widely used as a model system to study pathogenesis of these viruses in vivo. The effector of MHV68-induced host shutoff is its SOX homolog, here termed muSOX. There is remarkable functional conservation of muSOX host shutoff activities with those of KSHV SOX, including the recently described ability of SOX to induce mRNA hyperadenylation in the nucleus as well as cause nuclear relocalization of the poly(A) binding protein. SOX and muSOX localize to both the nucleus and cytoplasm of infected cells. Using spatially restricted variants of these proteins, we go on to demonstrate that all known host shutoff-related activities of SOX and muSOX are orchestrated exclusively from the cytoplasm. These results have important mechanistic implications for how SOX and muSOX target nascent cellular transcripts in the nucleus. Furthermore, our findings establish MHV68 as a new, genetically tractable model to study host shutoff.

How tails define the ending: divergent roles for polyadenylation in RNA stability and gene expression.

Polyadenylation in eukaryotes has traditionally been viewed as a means of stabilizing mRNAs and enhancing their translation. It is now appreciated, however, that there are multiple pathways to polyadenylation that can have opposing consequences for RNA stability and gene expression. The focus of this article will be on nuclear polyadenylation, highlighting the recent advances in our understanding of noncanonical polyadenylation events and how their outcomes contrast with those of normal mRNA polyadenylation. Much insight into these pathways derives from studies in yeast, although data from metazoans are now emerging which implicate widespread use of polyadenylation as a means of genetic regulation. We show herein that short upstream mRNA polyadenylation events occur in cells undergoing enhanced herpesvirus-mediated mRNA turnover, thereby extending the evidence for poly(A)-stimulated RNA decay in mammals.

Morphology and distribution of antennal sensilla in a parasitoid fly, Gymnosoma rotundatum (Diptera: Tachinidae).

Morphology of antennal sensilla and their distribution were investigated in male and female adults of the parasitoid fly Gymnosoma rotundatum (Diptera: Tachinidae) using scanning electron microscopy (SEM). The overall length and shape were not different between males and females from each other. Three basic types of sensilla (sensilla basiconica, s. chaetica, and s. coeloconica) were identified from both sexes, but with variations in numbers and distribution along the antennae. The s. basiconica and s. chaetica could be divided further into subtypes; s. basiconica into three subtypes and s. chaetica into two subtypes. All the basiconica subtypes 1, 2, and 3 were multiporous, indicating that their primary function was olfactory. The sensilla basiconica was most abundant on the antennae of both sexes. The abundance of s. basiconica subtype 1 was different, but other subtypes 2 and 3 were similar between males and females. There was no pore on the cuticular surface of the s. chaetica and s. coeloconica, suggesting that they are likely to be a mechanosensory or a thermohygroreceptory function. The abundance of the two sensillum types was similar between males and females. The morphological information obtained in our study provides a basis for future investigations into the sensory physiological function, and associated behaviors, of each type of sensilla in this parasitoid fly.

Future Needs for Science-Driven Geospatial and Temporal Extravehicular Activity Planning and Execution.

Future human missions to Mars are expected to emphasize scientific exploration. While recent Mars rover missions have addressed a wide range of science objectives, human extravehicular activities (EVAs), including the Apollo missions, have had limited experience with science operations. Current EVAs are carefully choreographed and guided continuously from Earth with negligible delay in communications between crew and flight controllers. Future crews on Mars will be expected to achieve their science objectives while operating and coordinating with a science team back on Earth under communication latency and bandwidth restrictions. The BASALT (Biologic Analog Science Associated with Lava Terrains) research program conducted Mars analog science on Earth to understand the concept of operations and capabilities needed to support these new kinds of EVAs. A suite of software tools (Minerva) was used for planning and executing all BASALT EVAs, supporting text communication across communication latency, and managing the collection of operational and scientific EVA data. This paper describes the support capabilities provided by Minerva to cope with various geospatial and temporal constraints to support the planning and execution phases of the EVAs performed during the BASALT research program. The results of this work provide insights on software needs for future science-driven planetary EVAs.

An Efficient Method for Electroporation of Small Interfering RNAs into ENCODE Project Tier 1 GM12878 and K562 Cell Lines.

The Encyclopedia of DNA Elements (ENCODE) Project aims to identify all functional sequence elements in the human genome sequence by use of high-throughput DNA/cDNA sequencing approaches. To aid the standardization, comparison, and integration of data sets produced from different technologies and platforms, the ENCODE Consortium selected several standard human cell lines to be used by the ENCODE Projects. The Tier 1 ENCODE cell lines include GM12878, K562, and H1 human embryonic stem cell lines. GM12878 is a lymphoblastoid cell line, transformed with the Epstein-Barr virus, that was selected by the International HapMap Project for whole genome and transcriptome sequencing by use of the Illumina platform. K562 is an immortalized myelogenous leukemia cell line. The GM12878 cell line is attractive for the ENCODE Projects, as it offers potential synergy with the International HapMap Project. Despite the vast amount of sequencing data available on the GM12878 cell line through the ENCODE Project, including transcriptome, chromatin immunoprecipitation-sequencing for histone marks, and transcription factors, no small interfering siRNA-mediated knockdown studies have been performed in the GM12878 cell line, as cationic lipid-mediated transfection methods are inefficient for lymphoid cell lines. Here, we present an efficient and reproducible method for transfection of a variety of siRNAs into the GM12878 and K562 cell lines, which subsequently results in targeted protein depletion.