Aberrant NOVA1 function disrupts alternative splicing in early stages of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a fatal disease characterized by aberrant alternative splicing (AS). Nuclear loss and cytoplasmic accumulation of the splicing factor TDP-43 in motor neurons (MN) are hallmarks of ALS at late stages of the disease. However, it is unknown if altered AS is present before TDP-43 pathology occurs. Here, we investigate altered AS and its origins in early stages of ALS using human induced pluripotent stem cell-derived motor neurons (MNs) from sporadic and familial ALS patients. We find high levels of the RNA-binding proteins NOVA1, NOVA2, and RBFOX2 in the insoluble protein fractions and observe that AS events in ALS-associated MNs are enriched for binding sites of these proteins. Our study points to an early disrupted function of NOVA1 that drives AS changes in a complex fashion, including events caused by a consistent loss of NOVA1 function. NOVA1 exhibits increased cytoplasmic protein levels in early stage MNs without TDP-43 pathology in ALS postmortem tissue. As nuclear TDP-43 protein level depletes, NOVA1 is reduced. Potential indications for a reduction of NOVA1 also came from mice over-expressing TDP-43 lacking its nuclear localization signal and iPSC-MN stressed with puromycin. This study highlights that additional RBP-RNA perturbations in ALS occur in parallel to TDP-43.

Discovery and functional interrogation of SARS-CoV-2 protein-RNA interactions.

The COVID-19 pandemic is caused by severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2). The betacoronvirus has a positive sense RNA genome which encodes for several RNA binding proteins. Here, we use enhanced crosslinking and immunoprecipitation to investigate SARS-CoV-2 protein interactions with viral and host RNAs in authentic virus-infected cells. SARS-CoV-2 proteins, NSP8, NSP12, and nucleocapsid display distinct preferences to specific regions in the RNA viral genome, providing evidence for their shared and separate roles in replication, transcription, and viral packaging. SARS-CoV-2 proteins expressed in human lung epithelial cells bind to 4773 unique host coding RNAs. Nine SARS-CoV-2 proteins upregulate target gene expression, including NSP12 and ORF9c, whose RNA substrates are associated with pathways in protein N-linked glycosylation ER processing and mitochondrial processes. Furthermore, siRNA knockdown of host genes targeted by viral proteins in human lung organoid cells identify potential antiviral host targets across different SARS-CoV-2 variants. Conversely, NSP9 inhibits host gene expression by blocking mRNA export and dampens cytokine productions, including interleukin-1α/ß. Our viral protein-RNA interactome provides a catalog of potential therapeutic targets and offers insight into the etiology of COVID-19 as a safeguard against future pandemics.

Discovery and functional interrogation of SARS-CoV-2 protein-RNA interactions.

The COVID-19 pandemic is caused by severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2). The betacoronvirus has a positive sense RNA genome which encodes for several RNA binding proteins. Here, we use enhanced crosslinking and immunoprecipitation to investigate SARS-CoV-2 protein interactions with viral and host RNAs in authentic virus-infected cells. SARS-CoV-2 proteins, NSP8, NSP12, and nucleocapsid display distinct preferences to specific regions in the RNA viral genome, providing evidence for their shared and separate roles in replication, transcription, and viral packaging. SARS-CoV-2 proteins expressed in human lung epithelial cells bind to 4773 unique host coding RNAs. Nine SARS-CoV-2 proteins upregulate target gene expression, including NSP12 and ORF9c, whose RNA substrates are associated with pathways in protein N-linked glycosylation ER processing and mitochondrial processes. Furthermore, siRNA knockdown of host genes targeted by viral proteins in human lung organoid cells identify potential antiviral host targets across different SARS-CoV-2 variants. Conversely, NSP9 inhibits host gene expression by blocking mRNA export and dampens cytokine productions, including interleukin-1α/ß. Our viral protein-RNA interactome provides a catalog of potential therapeutic targets and offers insight into the etiology of COVID-19 as a safeguard against future pandemics.

Huntington's disease mice and human brain tissue exhibit increased G3BP1 granules and TDP43 mislocalization.

Chronic cellular stress associated with neurodegenerative disease can result in the persistence of stress granule (SG) structures, membraneless organelles that form in response to cellular stress. In Huntington's disease (HD), chronic expression of mutant huntingtin generates various forms of cellular stress, including activation of the unfolded protein response and oxidative stress. However, it has yet to be determined whether SGs are a feature of HD neuropathology. We examined the miRNA composition of extracellular vesicles (EVs) present in the cerebrospinal fluid (CSF) of patients with HD and show that a subset of their target mRNAs were differentially expressed in the prefrontal cortex. Of these targets, SG components were enriched, including the SG-nucleating Ras GTPase-activating protein-binding protein 1 (G3BP1). We investigated localization and levels of G3BP1 and found a significant increase in the density of G3BP1-positive granules in the cortex and hippocampus of R6/2 transgenic mice and in the superior frontal cortex of the brains of patients with HD. Intriguingly, we also observed that the SG-associated TAR DNA-binding protein 43 (TDP43), a nuclear RNA/DNA binding protein, was mislocalized to the cytoplasm of G3BP1 granule-positive HD cortical neurons. These findings suggest that G3BP1 SG dynamics may play a role in the pathophysiology of HD.

Conserved metabolite regulation of stress granule assembly via AdoMet.

Stress granules (SGs) are evolutionarily conserved condensates of ribonucleoproteins that assemble in response to metabolic stresses. Because aberrant SG formation is associated with amyotrophic lateral sclerosis (ALS), understanding the connection between metabolic activity and SG composition can provide therapeutic insights into neurodegeneration. Here, we identify 17 metabolic enzymes recruited to yeast SGs in response to physiological growth stress. Furthermore, the product of one of these enzymes, AdoMet, is a regulator of SG assembly and composition. Decreases in AdoMet levels increase SG formation, while chronic elevation of AdoMet produces SG remnants lacking proteins associated with the 5' end of transcripts. Interestingly, acute elevation of AdoMet blocks SG formation in yeast and motor neurons. Treatment of ALS-derived motor neurons with AdoMet also suppresses the formation of TDP-43-positive SGs, a hallmark of ALS. Together, these results argue that AdoMet is an evolutionarily conserved regulator of SG composition and assembly with therapeutic potential in neurodegeneration.

Pooled CRISPR screens with imaging on microraft arrays reveals stress granule-regulatory factors.

Genetic screens using pooled CRISPR-based approaches are scalable and inexpensive, but restricted to standard readouts, including survival, proliferation and sortable markers. However, many biologically relevant cell states involve cellular and subcellular changes that are only accessible by microscopic visualization, and are currently impossible to screen with pooled methods. Here we combine pooled CRISPR-Cas9 screening with microraft array technology and high-content imaging to screen image-based phenotypes (CRaft-ID; CRISPR-based microRaft followed by guide RNA identification). By isolating microrafts that contain genetic clones harboring individual guide RNAs (gRNA), we identify RNA-binding proteins (RBPs) that influence the formation of stress granules, the punctate protein-RNA assemblies that form during stress. To automate hit identification, we developed a machine-learning model trained on nuclear morphology to remove unhealthy cells or imaging artifacts. In doing so, we identified and validated previously uncharacterized RBPs that modulate stress granule abundance, highlighting the applicability of our approach to facilitate image-based pooled CRISPR screens.

Untargeted mass spectrometry-based metabolomics approach unveils molecular changes in raw and processed foods and beverages.

In our daily lives, we consume foods that have been transported, stored, prepared, cooked, or otherwise processed by ourselves or others. Food storage and preparation have drastic effects on the chemical composition of foods. Untargeted mass spectrometry analysis of food samples has the potential to increase our chemical understanding of these processes by detecting a broad spectrum of chemicals. We performed a time-based analysis of the chemical changes in foods during common preparations, such as fermentation, brewing, and ripening, using untargeted mass spectrometry and molecular networking. The data analysis workflow presented implements an approach to study changes in food chemistry that can reveal global alterations in chemical profiles, identify changes in abundance, as well as identify specific chemicals and their transformation products. The data generated in this study are publicly available, enabling the replication and re-analysis of these data in isolation, and serve as a baseline dataset for future investigations.

Small-Molecule Modulation of TDP-43 Recruitment to Stress Granules Prevents Persistent TDP-43 Accumulation in ALS/FTD.

Stress granules (SGs) form during cellular stress and are implicated in neurodegenerative diseases such as amyotrophic lateral sclerosis and frontotemporal dementia (ALS/FTD). To yield insights into the role of SGs in pathophysiology, we performed a high-content screen to identify small molecules that alter SG properties in proliferative cells and human iPSC-derived motor neurons (iPS-MNs). One major class of active molecules contained extended planar aromatic moieties, suggesting a potential to intercalate in nucleic acids. Accordingly, we show that several hit compounds can prevent the RNA-dependent recruitment of the ALS-associated RNA-binding proteins (RBPs) TDP-43, FUS, and HNRNPA2B1 into SGs. We further demonstrate that transient SG formation contributes to persistent accumulation of TDP-43 into cytoplasmic puncta and that our hit compounds can reduce this accumulation in iPS-MNs from ALS patients. We propose that compounds with planar moieties represent a promising starting point to develop small-molecule therapeutics for treating ALS/FTD.

Transcriptome-pathology correlation identifies interplay between TDP-43 and the expression of its kinase CK1E in sporadic ALS.

Sporadic amyotrophic lateral sclerosis (sALS) is the most common form of ALS, however, the molecular mechanisms underlying cellular damage and motor neuron degeneration remain elusive. To identify molecular signatures of sALS we performed genome-wide expression profiling in laser capture microdissection-enriched surviving motor neurons (MNs) from lumbar spinal cords of sALS patients with rostral onset and caudal progression. After correcting for immunological background, we discover a highly specific gene expression signature for sALS that is associated with phosphorylated TDP-43 (pTDP-43) pathology. Transcriptome-pathology correlation identified casein kinase 1ε (CSNK1E) mRNA as tightly correlated to levels of pTDP-43 in sALS patients. Enhanced crosslinking and immunoprecipitation in human sALS patient- and healthy control-derived frontal cortex, revealed that TDP-43 binds directly to and regulates the expression of CSNK1E mRNA. Additionally, we were able to show that pTDP-43 itself binds RNA. CK1E, the protein product of CSNK1E, in turn interacts with TDP-43 and promotes cytoplasmic accumulation of pTDP-43 in human stem-cell-derived MNs. Pathological TDP-43 phosphorylation is therefore, reciprocally regulated by CK1E activity and TDP-43 RNA binding. Our framework of transcriptome-pathology correlations identifies candidate genes with relevance to novel mechanisms of neurodegeneration.

Distinct and shared functions of ALS-associated proteins TDP-43, FUS and TAF15 revealed by multisystem analyses.

The RNA-binding protein (RBP) TAF15 is implicated in amyotrophic lateral sclerosis (ALS). To compare TAF15 function to that of two ALS-associated RBPs, FUS and TDP-43, we integrate CLIP-seq and RNA Bind-N-Seq technologies, and show that TAF15 binds to ∼4,900 RNAs enriched for GGUA motifs in adult mouse brains. TAF15 and FUS exhibit similar binding patterns in introns, are enriched in 3' untranslated regions and alter genes distinct from TDP-43. However, unlike FUS and TDP-43, TAF15 has a minimal role in alternative splicing. In human neural progenitors, TAF15 and FUS affect turnover of their RNA targets. In human stem cell-derived motor neurons, the RNA profile associated with concomitant loss of both TAF15 and FUS resembles that observed in the presence of the ALS-associated mutation FUS R521G, but contrasts with late-stage sporadic ALS patients. Taken together, our findings reveal convergent and divergent roles for FUS, TAF15 and TDP-43 in RNA metabolism.

Divergent roles of ALS-linked proteins FUS/TLS and TDP-43 intersect in processing long pre-mRNAs.

FUS/TLS (fused in sarcoma/translocated in liposarcoma) and TDP-43 are integrally involved in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia. We found that FUS/TLS binds to RNAs from >5,500 genes in mouse and human brain, primarily through a GUGGU-binding motif. We identified a sawtooth-like binding pattern, consistent with co-transcriptional deposition of FUS/TLS. Depletion of FUS/TLS from the adult nervous system altered the levels or splicing of >950 mRNAs, most of which are distinct from RNAs dependent on TDP-43. Abundance of only 45 RNAs was reduced after depletion of either TDP-43 or FUS/TLS from mouse brain, but among these were mRNAs that were transcribed from genes with exceptionally long introns and that encode proteins that are essential for neuronal integrity. Expression levels of a subset of these were lowered after TDP-43 or FUS/TLS depletion in stem cell-derived human neurons and in TDP-43 aggregate-containing motor neurons in sporadic ALS, supporting a common loss-of-function pathway as one component underlying motor neuron death from misregulation of TDP-43 or FUS/TLS.

LIN28 binds messenger RNAs at GGAGA motifs and regulates splicing factor abundance.

LIN28 is a conserved RNA-binding protein implicated in pluripotency, reprogramming, and oncogenesis. It was previously shown to act primarily by blocking let-7 microRNA (miRNA) biogenesis, but here we elucidate distinct roles of LIN28 regulation via its direct messenger RNA (mRNA) targets. Through crosslinking and immunoprecipitation coupled with high-throughput sequencing (CLIP-seq) in human embryonic stem cells and somatic cells expressing exogenous LIN28, we have defined discrete LIN28-binding sites in a quarter of human transcripts. These sites revealed that LIN28 binds to GGAGA sequences enriched within loop structures in mRNAs, reminiscent of its interaction with let-7 miRNA precursors. Among LIN28 mRNA targets, we found evidence for LIN28 autoregulation and also direct but differing effects on the protein abundance of splicing regulators in somatic and pluripotent stem cells. Splicing-sensitive microarrays demonstrated that exogenous LIN28 expression causes widespread downstream alternative splicing changes. These findings identify important regulatory functions of LIN28 via direct mRNA interactions.

Integrative genome-wide analysis reveals cooperative regulation of alternative splicing by hnRNP proteins.

Understanding how RNA binding proteins control the splicing code is fundamental to human biology and disease. Here, we present a comprehensive study to elucidate how heterogeneous nuclear ribonucleoparticle (hnRNP) proteins, among the most abundant RNA binding proteins, coordinate to regulate alternative pre-mRNA splicing (AS) in human cells. Using splicing-sensitive microarrays, crosslinking and immunoprecipitation coupled with high-throughput sequencing (CLIP-seq), and cDNA sequencing, we find that more than half of all AS events are regulated by multiple hnRNP proteins and that some combinations of hnRNP proteins exhibit significant synergy, whereas others act antagonistically. Our analyses reveal position-dependent RNA splicing maps, in vivo consensus binding sites, a surprising level of cross- and autoregulation among hnRNP proteins, and the coordinated regulation by hnRNP proteins of dozens of other RNA binding proteins and genes associated with cancer. Our findings define an unprecedented degree of complexity and compensatory relationships among hnRNP proteins and their splicing targets that likely confer robustness to cells.