The RNA-binding landscape of HAX1 protein indicates its involvement in ribosome biogenesis and translation.

HAX1 is a human protein with no known homologues or structural domains, mutations in which cause severe congenital neutropenia through mechanisms that are poorly understood. Previous studies reported RNA-binding capacity of HAX1, but the role of this binding in physiology and pathology remains unexplained. Here we report transcriptome-wide characterization of HAX1 RNA targets using RIP-seq and CRAC, indicating that HAX1 binds transcripts involved in ribosome biogenesis and rRNA processing. Using CRISPR knockouts we find that RNA targets of HAX1 partially overlap with transcripts downregulated in HAX1 KO, implying a role in mRNA stabilization. Gene ontology analysis demonstrated that genes differentially expressed in HAX1 KO (including genes involved in ribosome biogenesis and translation) are also enriched in a subset of genes whose expression correlates with HAX1 expression in four analyzed neoplasms. Functional connection to ribosome biogenesis was also demonstrated by gradient sedimentation ribosome profiles, which revealed differences in the small subunit:monosome ratio in HAX1 WT/KO. We speculate that changes in HAX1 expression may be important for the etiology of HAX1-linked diseases through dysregulation of translation.

Degradome comparison between wild and cultivated rice identifies differential targeting by miRNAs

Background Small non-coding (s)RNAs are involved in the negative regulation of gene expression, playing critical roles in genome integrity, development and metabolic pathways. Targeting of RNAs by ribonucleoprotein complexes of sRNAs bound to Argonaute (AGO) proteins results in cleaved RNAs having precise and predictable 5` ends. While tools to study sliced bits of RNAs to confirm the efficiency of sRNA-mediated regulation are available, they are sub-optimal. In this study, we provide an improvised version of a tool with better efficiency to accurately validate sRNA targets. Results Here, we improvised the CleaveLand tool to identify additional micro (mi)RNA targets that belong to the same family and also other targets within a specified free energy cut-off. These additional targets were otherwise excluded during the default run. We employed these tools to understand the sRNA targeting efficiency in wild and cultivated rice, sequenced degradome from two rice lines, O. nivara and O. sativa indica Pusa Basmati-1 and analyzed variations in sRNA targeting. Our results indicate the existence of multiple miRNA-mediated targeting differences between domesticated and wild species. For example, Os5NG4 was targeted only in wild rice that might be responsible for the poor secondary wall formation when compared to cultivated rice. We also identified differential mRNA targets of secondary sRNAs that were generated after miRNA-mediated cleavage of primary targets. Conclusions We identified many differentially targeted mRNAs between wild and domesticated rice lines. In addition to providing a step-wise guide to generate and analyze degradome datasets, we showed how domestication altered sRNA-mediated cascade silencing during the evolution of indica rice.

RNA binding protein HuD promotes autophagy and tumor stress survival by suppressing mTORC1 activity and augmenting ARL6IP1 levels

Background Neuronal-origin HuD (ELAVL4) is an RNA binding protein overexpressed in neuroblastoma (NB) and certain other cancers. The RNA targets of this RNA binding protein in neuroblastoma cells and their role in promoting cancer survival have been unexplored. In the study of modulators of mTORC1 activity under the conditions of optimal cell growth and starvation, the role of HuD and its two substrates were studied. Methods RNA immunoprecipitation/sequencing (RIP-SEQ) coupled with quantitative real-time PCR were used to identify substrates of HuD in NB cells. Validation of the two RNA targets of HuD was via reverse capture of HuD by synthetic RNA oligoes from cell lysates and binding of RNA to recombinant forms of HuD in the cell and outside of the cell. Further analysis was via RNA transcriptome analysis of HuD silencing in the test cells. Results In response to stress, HuD was found to dampen mTORC1 activity and allow the cell to upregulate its autophagy levels by suppressing mTORC1 activity. Among mRNA substrates regulated cell-wide by HuD, GRB-10 and ARL6IP1 were found to carry out critical functions for survival of the cells under stress. GRB-10 was involved in blocking mTORC1 activity by disrupting Raptor-mTOR kinase interaction. Reduced mTORC1 activity allowed lifting of autophagy levels in the cells required for increased survival. In addition, ARL6IP1, an apoptotic regulator in the ER membrane, was found to promote cell survival by negative regulation of apoptosis. As a therapeutic target, knockdown of HuD in two xenograft models of NB led to a block in tumor growth, confirming its importance for viability of the tumor cells. Cell-wide RNA messages of these two HuD substrates and HuD and mTORC1 marker of activity significantly correlated in NB patient populations and in mouse xenografts. Conclusions HuD is seen as a novel means of promoting stress survival in this cancer type by downregulating mTORC1 activity and negatively regulating apoptosis.

Persistence of endogenous SARS-CoV-2 and pepper mild mottle virus RNA in wastewater settled solids

Limited information is available on the decay rate of endogenous SARS-CoV-2 and pepper mild mottle virus (PMMoV) RNA in wastewater and primary settled solids, potentially limiting an understanding of how transit or holding times within wastewater infrastructure might impact RNA measurements and their relationship to community COVID-19 infections. In this study, primary settled solids samples were collected from two wastewater treatment plants in the San Francisco Bay Area. Samples were thoroughly mixed, aliquoted into subsamples, and stored at 4°C, 22°C, and 37°C for 10 days. The concentration of SARS-CoV-2 (N1 and N2 targets) and PMMoV RNA was measured using an RT-ddPCR. Limited decay (< 1 log10 reduction) was observed in the detection of viral RNA targets at all temperature conditions, suggesting that SARS-CoV-2 and PMMoV RNA can be highly persistent in solids. First-order decay rate constants ranged from 0.011 - 0.098 day-1 for SARS-CoV-2 RNA and 0.010 - 0.091 day-1 for PMMoV RNA, depending on temperature conditions. Slower decay was observed for SARS-CoV-2 RNA in primary settled solids compared to previously reported decay in wastewater influent. Further research is needed to understand if solid content and wastewater characteristics might influence the persistence of viral RNA targets.

The lncRNAs at X Chromosome Inactivation Center: Not Just a Matter of Sex Dosage Compensation

Non-coding RNAs (ncRNAs) constitute the majority of the transcriptome, as the result of pervasive transcription of the mammalian genome. Different RNA species, such as lncRNAs, miRNAs, circRNA, mRNAs, engage in regulatory networks based on their reciprocal interactions, often in a competitive manner, in a way denominated “competing endogenous RNA (ceRNA) networks” (“ceRNET”): miRNAs and other ncRNAs modulate each other, since miRNAs can regulate the expression of lncRNAs, which in turn regulate miRNAs, titrating their availability and thus competing with the binding to other RNA targets. The unbalancing of any network component can derail the entire regulatory circuit acting as a driving force for human diseases, thus assigning “new” functions to “old” molecules. This is the case of XIST, the lncRNA characterized in the early 1990s and well known as the essential molecule for X chromosome inactivation in mammalian females, thus preventing an imbalance of X-linked gene expression between females and males. Currently, literature concerning XIST biology is becoming dominated by miRNA associations and they are also gaining prominence for other lncRNAs produced by the X-inactivation center. This review discusses the available literature to explore possible novel functions related to ceRNA activity of lncRNAs produced by the X-inactivation center, beyond their role in dosage compensation, with prospective implications for emerging gender-biased functions and pathological mechanisms.

Human neural networks with sparse TDP-43 pathology reveal NPTX2 misregulation in ALS/FTLD

Human cellular models of neurodegeneration require reproducibility and longevity, which is necessary for simulating these age-dependent diseases. Such systems are particularly needed for TDP-43 proteinopathies, which involve human-specific mechanisms that cannot be directly studied in animal models. To explore the emergence and consequences of TDP-43 pathologies, we generated iPSC-derived, colony morphology neural stem cells (iCoMoNSCs) via manual selection of neural precursors. Single-cell transcriptomics (scRNA-seq) and comparison to independent NSCs, showed that iCoMoNSCs are uniquely homogenous and self-renewing. Differentiated iCoMoNSCs formed a self-organized multicellular system consisting of synaptically connected and electrophysiologically active neurons, which matured into long-lived functional networks. Neuronal and glial maturation in iCoMoNSC-derived cultures was similar to that of cortical organoids. Overexpression of wild-type TDP-43 in a minority of iCoMoNSC-derived neurons led to progressive fragmentation and aggregation, resulting in loss of function and neurotoxicity. scRNA-seq revealed a novel set of misregulated RNA targets coinciding in both TDP-43 overexpressing neurons and patient brains exhibiting loss of nuclear TDP-43. The strongest misregulated target encoded for the synaptic protein NPTX2, which was consistently misaccumulated in ALS and FTLD patient neurons with TDP-43 pathology. Our work directly links TDP-43 misregulation and NPTX2 accumulation, thereby highlighting a new pathway of neurotoxicity.

Tailer: A Pipeline for Sequencing-Based Analysis of Non-Polyadenylated RNA 3’ End Processing

Post-transcriptional trimming and tailing of RNA 3’ ends play key roles in the processing and quality control of non-coding RNAs (ncRNAs). However, bioinformatic tools to examine changes in the RNA 3’ “tailome” are sparse and not standardized. Here we present Tailer, a bioinformatic pipeline in two parts that allows for robust quantification and analysis of tail information from next generation sequencing experiments that preserve RNA 3’ end information. The first part of Tailer, Tailer-Processing, uses genome annotation or reference FASTA gene sequences to quantify RNA 3’ ends from SAM-formatted alignment files or FASTQ sequence read files produced from sequencing experiments. The second part, Tailer-Analysis, uses the output of Tailer-Processing to identify statistically significant RNA targets of trimming and tailing and create graphs for data exploration. We apply Tailer to RNA 3’ end sequencing experiments from three published studies and find that it accurately and reproducibly recapitulates key findings. Thus, Tailer should be a useful and easily accessible tool to globally investigate tailing dynamics of non-polyadenylated RNAs and conditions that perturb them.

RNA Binding Protein SRSF3 confers an essential role in megakaryocyte maturation and platelet production

RNA processing is increasingly recognised as a critical control point in the regulation of different haematopoietic lineages including megakaryocytes responsible for the production of platelets. Platelets are anucleate cytoplasts that contain a rich repertoire of RNAs encoding proteins with essential platelet functions derived from the parent megakaryocyte. It is largely unknown how RNA binding proteins contribute to the development and functions of megakaryocytes and platelets. We show that Serine-arginine rich splicing factor 3 (SRSF3) is essential for megakaryocyte maturation and generation of functional platelets. Megakaryocyte-specific deletion of Srsf3 in mice led to macrothrombocytopenia characterised by megakaryocyte maturation arrest, dramatically reduced platelet counts and abnormally large functionally compromised platelets. SRSF3 deficient megakaryocytes failed to reprogram their transcriptome during maturation and to load platelets with RNAs required for normal platelet function. SRSF3 depletion led to nuclear accumulation of megakaryocyte mRNAs demonstrating that SRSF3 deploys similar RNA regulatory mechanisms in megakaryocytes as in other cell types. Our study further suggests that SRSF3 plays a role in sorting cytoplasmic megakaryocyte RNAs into platelets and demonstrates how SRSF3-mediated RNA processing forms a central part of megakaryocyte gene regulation. Understanding SRSF3 functions in megakaryocytes and platelets provides key insights into normal thrombopoiesis and platelet pathologies as SRSF3 RNA targets in megakaryocytes are associated with platelet diseases.

The RNA-binding protein Orb2 is associated with microcephaly and supports centrosome asymmetry in neural stem cells

To maintain a balance of self-renewal versus neurogenesis, neural stem cells (NSCs) undergo repeated cycles of asymmetric cell division along an invariant polarity axis instructed by centrosomes. During interphase, the NSC centrosomes are defined by marked asymmetries in protein composition and functional activity as microtubule-organizing centers. Here, we show a conserved RNA-binding protein, Orb2, supports centrosome asymmetry in interphase NSCs. While Orb2 localizes to the active apical centrosome, it promotes the transient inactivation of the basal centrosome required for centrosome segregation and spindle morphogenesis. Orb2 is required cell autonomously within NSCs to support centrosome asymmetry and maintenance of the stem cell pool. Conversely, loss of orb2 manifests in microcephaly independent of Orb2 function in NSCs. We suggest Orb2 plays opposing roles in centrosome activation and inactivation, possibly through the translational regulation of multiple mRNA substrates. Bioinformatics uncovers a significant overlap among RNA targets between Drosophila Orb2 and human CPEB4, consistent with a conserved role for CPEB proteins in in centrosome regulation and neurodevelopment.