RNA Splicing Protein Research Articles

Reclassification of Two MLH1 Variants of Uncertain Significance Utilizing Clinical and Functional Data.

Pathogenic variants in the mismatch repair genes are associated with an elevated lifetime risk of colorectal cancer (CRC). We previously identified two variants of uncertain significance (VUS) in the MLH1 gene, c.696_698del, p.(Cys233del) and c.1919C > G, p.(Pro640Arg), in Danish families with numerous occurrences of CRC. To reclassify the variants we collected clinical data, initiated tumor and co-segregation analysis, and performed RNA splicing analysis, subcellular localization, and protein stability studies. The functional analysis revealed that the c.696_698del, p.(Cys233del) variant had an effect at the RNA level, on subcellular localization, and on protein stability, while the c.1919C > G, p.(Pro640Arg) variant showed decreased expression in localization studies and decreased protein stability. These results suggest both variants disrupt DNA mismatch repair. By applying all collected data and functional results we propose to reclassify the c.696_698del, p.(Cys233del) and the c.1919C > G, p.(Pro640Arg) variants as likely pathogenic (class 4) using MMR gene-specific ACMG/AMP guidelines. Consequently, the two MLH1 variants can now be used for risk assessment of variant carriers, while family members without the variants can be excluded from intensified cancer surveillance and follow population recommendations.

BCL11b interacts with RNA and proteins involved in RNA processing and developmental diseases

BCL11b is a transcription regulator and a tumor suppressor involved in lymphomagenesis, central nervous system (CNS) and immune system developments. BCL11b favors persistence of HIV latency and contributes to control cell cycle, differentiation and apoptosis in multiple organisms and cell models. Although BCL11b recruits the non-coding RNA 7SK and epigenetic enzymes to regulate gene expression, BCL11b-associated ribonucleoprotein complexes are unknown. Thanks to CLIP-seq and quantitative LC-MS/MS mass spectrometry approaches complemented with systems biology validations, we show that BCL11b interacts with RNA splicing and non-sense-mediated decay proteins, including FUS, SMN1, UPF1 and Drosha, which may contribute in isoform selection of protein-coding RNA isoforms from noncoding-RNAs isoforms (retained introns or nonsense mediated RNA). Interestingly, BCL11b binds to RNA transcripts and proteins encoded by the same genes (FUS, ESWR1, CHD and Tubulin). Our study highlights that BCL11b targets RNA processing and splicing proteins, and RNAs that implicate cell cycle, development, neurodegenerative, and cancer pathways. These findings will help future mechanistic understanding of developmental disorders. ImportanceBCL11b-protein and RNA interactomes reveal BLC11b association with specific nucleoprotein complexes involved in the regulation of genes expression.BCL11b interacts with RNA processing and splicing proteins.

Dehydration priming remodels protein abundance and phosphorylation level regulating tolerance to subsequent dehydration or salt stress in creeping bentgrass

Dehydration priming (DP) induces stress memory which plays a positive role in plant adaptability, but it is not well understood how DP differentially regulates subsequent dehydration (cis priming) or salt (trans priming) tolerance at the post-translational level. Purpose of this study was to identify proteins, phosphorylation levels and sites, and relevant metabolic pathways for DP-induced dehydration or salt tolerance in Agrostis stolonifera. DP-induced differentially regulated proteins (DRPs) were mostly located in the cytoplasm, chloroplast, and cell membrane, and differentially regulated phosphoproteins (DRPPs) were mostly nuclear proteins and cytoplasmic proteins. DP regulated common phosphorylation sites ([SP] and [RxxS]) under dehydration and salt conditions and also individually affected 8 or 11 phosphorylation sites under dehydration or salt stress. DP-regulated DRPPs were mainly rich in glycolysis and glutathione metabolism pathways, RNA splicing, and dynamin family proteins under dehydration stress, whereas DP-regulated salt tolerance was mainly related to chlorophyll metabolism, photosynthesis, MAPK signaling cascade, and ABC transporter I family at the phosphorylation level. In addition, the DP also significantly up-regulated phosphorylation of histones (ATXR3 and SETD1A) in response to subsequent dehydration and salt stress as well as abundances of antioxidant enzymes, dynamin family protein, and KCS6 under dehydration stress or abundances of PETE, HMGA, XTH, and ABCI6 under salt stress, respectively. Transcriptomics analysis further indicated that DP-regulated dehydration or salt tolerance was also related to transcriptional regulation in the early stage. Current results provided better understanding of the role of stress memory in plant adaptability to repeated or crossed stress via post-translational modifications (PTMs). SignificanceRecurrent moderate drought may buffer drought legacies in many plant species. When plants were exposed to repeated drought stress, their adaptability to subsequent stress could be enhanced, which is known as “stress memory”. Dehydration priming has been found to be an important approach to induce stress memory. Current results provided better understanding of the role of stress memory in plant adaptability to repeated or crossed stress via post-translational modifications.

SON is an essential RNA splicing factor promoting ErbB2 and ErbB3 expression in breast cancer.

In breast cancer, ErbB receptors play a critical role, and overcoming drug resistance remains a major challenge in the clinic. However, intricate regulatory mechanisms of ErbB family genes are poorly understood. Here, we demonstrate SON as an ErbB-regulatory splicing factor and a novel therapeutic target for ErbB-positive breast cancer. SON and ErbB expression analyses using public database, patient tissue microarray, and cell lines were performed. SON knockdown assessed its impact on cell proliferation, apoptosis, kinase phosphorylation, RNA splicing, and in vivo tumour growth. RNA immunoprecipitation was performed to measure SON binding. SON is highly expressed in ErbB2-positive breast cancer patient samples, inversely correlating with patient survival. SON knockdown induced intron retention in selective splice sites within ErbB2 and ErbB3 transcripts, impairing effective RNA splicing and reducing protein expression. SON disruption suppressed downstream kinase signalling of ErbB2/3, including the Akt, p38, and JNK pathways, with increased vulnerability in ErbB2-positive breast cancer cells compared to ErbB2-negative cells. SON silencing in ErbB2-positive breast cancer xenografts led to tumour regression in vivo. We identified SON as a novel RNA splicing factor that plays a critical role in regulating ErbB2/3 expression, suggesting SON is an ideal therapeutic target in ErbB2-positive breast cancers.

MRS2 missense variation at Asp216 abrogates inhibitory Mg2+ binding, potentiating cell migration and apoptosis resistance.

Mitochondrial magnesium (Mg2+) is a crucial modulator of protein stability, enzymatic activity, ATP synthesis, and cell death. Mitochondrial RNA splicing protein 2 (MRS2) is the main Mg2+ channel in the inner mitochondrial membrane that mediates influx into the matrix. Recent cryo-electron microscopy (cryo-EM) human MRS2 structures exhibit minimal conformational changes at high and low Mg2+, yet the regulation of human MRS2 and orthologues by Mg2+ binding to analogous matrix domains has been well established. Further, a missense variation at D216 has been identified associated with malignant melanoma and MRS2 expression and activity is implicated in gastric cancer. Thus, to gain more mechanistic and functional insight into Mg2+ sensing by the human MRS2 matrix domain and the association with proliferative disease, we assessed the structural, biophysical, and functional effects of a D216Q mutant. We show that the D216Q mutation is sufficient to abrogate Mg2+-binding and associated conformational changes including increased α-helicity, stability, and monomerization. Further, we reveal that the MRS2 matrix domains interact with ~μM affinity, which is weakened by up to two orders of magnitude in the presence of Mg2+ for wild-type but unaffected for D216Q. Finally, we demonstrate the importance of Mg2+ sensing by MRS2 to prevent matrix Mg2+ overload as HeLa cells overexpressing MRS2 show enhanced Mg2+ uptake, cell migration, and resistance to apoptosis while MRS2 D216Q robustly potentiates these cancer phenotypes. Collectively, our findings further define the MRS2 matrix domain as a critical Mg2+ sensor that undergoes conformational and assembly changes upon Mg2+ interactions dependent on D216 to temper matrix Mg2+ overload.

The Rice YL4 Gene Encoding a Ribosome Maturation Domain Protein Is Essential for Chloroplast Development.

Chloroplast RNA splicing and ribosome maturation (CRM) domain proteins are a family of plant-specific proteins associated with RNA binding. In this study, we have conducted a detailed characterization of a novel rice CRM gene (LOC_Os04g39060) mutant, yl4, which showed yellow-green leaves at all the stages, had fewer tillers, and had a decreased plant height. Map-based cloning and CRISPR/Cas9 editing techniques all showed that YL4 encoded a CRM domain protein in rice. In addition, subcellular localization revealed that YL4 was in chloroplasts. YL4 transcripts were highly expressed in all leaves and undetectable in roots and stems, and the mutation of YL4 affected the transcription of chloroplast-development-related genes. This study indicated that YL4 is essential for chloroplast development and affects some agronomic traits.

In silico identification of a novel Cdc2-like kinase 2 (CLK2) inhibitor in triple negative breast cancer.

Dysregulation of RNA splicing processes is intricately linked to tumorigenesis in various cancers, especially breast cancer. Cdc2-like kinase 2 (CLK2), an oncogenic RNA-splicing kinase pivotal in breast cancer, plays a significant role, particularly in the context of triple-negative breast cancer (TNBC), a subtype marked by substantial medical challenges due to its low survival rates. In this study, we employed a structure-based virtual screening (SBVS) method to identify potential CLK2 inhibitors with novel chemical structures for treating TNBC. Compound 670551 emerged as a novel CLK2 inhibitor with a 50% inhibitory concentration (IC50) value of 619.7 nM. Importantly, Compound 670551 exhibited high selectivity for CLK2 over other protein kinases. Functionally, this compound significantly reduced the survival and proliferation of TNBC cells. Results from a cell-based assay demonstrated that this inhibitor led to a decrease in RNA splicing proteins, such as SRSF4 and SRSF6, resulting in cell apoptosis. In summary, we identified a novel CLK2 inhibitor as a promising potential treatment for TNBC therapy.

Mapping the daily rhythmic transcriptome in the diabetic retina

Retinal function changes dramatically from day to night, yet clinical diagnosis, treatments, and experimental sampling occur during the day. To begin to address this gap in our understanding of disease pathobiology, this study investigates whether diabetes affects the retina's daily rhythm of gene expression. Diabetic, Ins2Akita/J mice, and non-diabetic littermates were kept under a 12 h:12 h light/dark cycle until 4 months of age. mRNA sequencing was conducted in retinas collected every 4 h throughout the 24 hr light/dark cycle. Computational approaches were used to detect rhythmicity, predict acrophase, identify differential rhythmic patterns, analyze phase set enrichment, and predict upstream regulators. The retinal transcriptome exhibited a tightly regulated rhythmic expression with a clear 12-hr transcriptional axis. Day-peaking genes were enriched for DNA repair, RNA splicing, and ribosomal protein synthesis, night-peaking genes for metabolic processes and growth factor signaling. Although the 12-hr transcriptional axis is retained in the diabetic retina, it is phase advanced for some genes. Upstream regulator analysis for the phase-shifted genes identified oxygen-sensing mechanisms and HIF1alpha, but not the circadian clock, which remained in phase with the light/dark cycle. We propose a model in which, early in diabetes, the retina is subjected to an internal desynchrony with the circadian clock and its outputs are still light-entrained whereas metabolic pathways related to neuronal dysfunction and hypoxia are phase advanced. Further studies are now required to evaluate the chronic implications of such desynchronization on the development of diabetic retinopathy.

Hypothermic Protection in Neocortex Is Topographic and Laminar, Seizure Unmitigating, and Partially Rescues Neurons Depleted of RNA Splicing Protein Rbfox3/NeuN in Neonatal Hypoxic-Ischemic Male Piglets.

The effects of hypothermia on neonatal encephalopathy may vary topographically and cytopathologically in the neocortex with manifestations potentially influenced by seizures that alter the severity, distribution, and type of neuropathology. We developed a neonatal piglet survival model of hypoxic-ischemic (HI) encephalopathy and hypothermia (HT) with continuous electroencephalography (cEEG) for seizures. Neonatal male piglets received HI-normothermia (NT), HI-HT, sham-NT, or sham-HT treatments. Randomized unmedicated sham and HI piglets underwent cEEG during recovery. Survival was 2-7 days. Normal and pathological neurons were counted in different neocortical areas, identified by cytoarchitecture and connectomics, using hematoxylin and eosin staining and immunohistochemistry for RNA-binding FOX-1 homolog 3 (Rbfox3/NeuN). Seizure burden was determined. HI-NT piglets had a reduced normal/total neuron ratio and increased ischemic-necrotic/total neuron ratio relative to sham-NT and sham-HT piglets with differing severities in the anterior and posterior motor, somatosensory, and frontal cortices. Neocortical neuropathology was attenuated by HT. HT protection was prominent in layer III of the inferior parietal cortex. Rbfox3 immunoreactivity distinguished cortical neurons as: Rbfox3-positive/normal, Rbfox3-positive/ischemic-necrotic, and Rbfox3-depleted. HI piglets had an increased Rbfox3-depleted/total neuron ratio in layers II and III compared to sham-NT piglets. Neuronal Rbfox3 depletion was partly rescued by HT. Seizure burdens in HI-NT and HI-HT piglets were similar. We conclude that the neonatal HI piglet neocortex has: (1) suprasylvian vulnerability to HI and seizures; (2) a limited neuronal cytopathological repertoire in functionally different regions that engages protective mechanisms with HT; (3) higher seizure burden, insensitive to HT, that is correlated with more panlaminar ischemic-necrotic neurons in the somatosensory cortex; and (4) pathological RNA splicing protein nuclear depletion that is sensitive to HT. This work demonstrates that HT protection of the neocortex in neonatal HI is topographic and laminar, seizure unmitigating, and restores neuronal depletion of RNA splicing factor.

Abstract B045: Evaluating the role of altered RNA metabolism in pancreatic cancer therapy resistance

Abstract Pancreatic ductal adenocarcinomas (PDACs) are highly lethal, mostly because they quickly develop resistance to current chemotherapies: Gemcitabine (GEM)/paclitaxel or cocktail FOLFIRINOX. Both chemotherapies rely on nucleoside analogues (GEM and 5-fluorouracil [5-FU], and resistance is developed without acquiring mutations, suggesting the role of non-mutational mechanisms for therapy resistance. Our recent findings demonstrated that PDACs depend on RNA splicing proteins to maintain tumors and are exquisitely susceptible to a range of therapies directed at RNA splicing, supporting the role of aberrant RNA splicing in PDAC therapy resistance. Here we evaluated the role of altered RNA splicing in PDAC therapy resistance. Three murine and human isogenic lines with either sensitivity or resistance to GEM were developed. We performed deep RNA sequencing (RNASeq, 80-100 million reads, traditional is 20-40 million reads), differential gene expression, and splicing analyses. GEM resistant PDAC cells demonstrated >40% of non-canonical RNA splice variants and altered expression of 30% of RNA-binding proteins encoded in the human/murine genome, compared to GEM sensitive ones. Specific alternatively spliced exons with common cis-elements were identified in therapy resistant cells, suggesting that these splicing changes are guided by a splicing factor or other RNA-binding protein(s) (RBPs). Top splicing changes were associated to mRNAs encoding ether lipid and pyruvate metabolism. This finding is important as previous studies have linked these metabolic pathways to therapy resistance, however, they have overlooked if protein isoforms, resulting from aberrant splicing, have different impacts in therapy resistance. In addition, GEM and H3B-8800, a novel spliceosome inhibitor showed higher synergistic activity in GEM resistant PDAC cells compared to GEM sensitive cells, suggesting that the development of resistance in PDAC is highly dependent on RNA splicing. Combinedly, these data strongly suggest that resistance to chemotherapeutic agents in PDAC cells requires altered RNA splicing and aberrant expression of associated proteins. Further investigation is ongoing to identify the splicing events and RBPs related to chemotherapy resistance, which will uncover novel mechanisms of therapy resistance and therapeutic modalities by targeting RNA splicing in pancreatic cancer. Citation Format: Md Afjalus Siraj, Xinning Shan, Robert Tseng, Luisa Escobar-Hoyos. Evaluating the role of altered RNA metabolism in pancreatic cancer therapy resistance [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr B045.

Srsf2P95H/+ co-operates with loss of TET2 to promote myeloid bias and initiate a chronic myelomonocytic leukemia-like disease in mice.

Recurrent mutations in RNA splicing proteins and epigenetic regulators contribute to the development of myelodysplastic syndrome (MDS) and related myeloid neoplasms. In chronic myelomonocytic leukemia (CMML), SRSF2 mutations occur in ~50% of patients and TET2 mutations in ~60%. Clonal analysis indicates that either mutation can arise as the founder lesion. Based on human cancer genetics we crossed an inducible Srsf2P95H/+ mutant model with Tet2fl/fl mice to mutate both concomitantly in hematopoietic stem cells. At 20-24 weeks post mutation induction, we observed subtle differences in the Srsf2/Tet2 mutants compared to either single mutant. Under conditions of native hematopoiesis with aging, we see a distinct myeloid bias and monocytosis in the Srsf2/Tet2 mutants. A subset of the compound Srsf2/Tet2 mutants display an increased granulocytic and distinctive monocytic proliferation (myelomonocytic hyperplasia), with increased immature promonocytes and monoblasts and binucleate promonocytes. Exome analysis of progressed disease demonstrated mutations in genes and pathways similar to those reported in human CMML. Upon transplantation, recipients developed leukocytosis, monocytosis, and splenomegaly. We reproduce Srsf2/Tet2 co-operativity in vivo, yielding a disease with core characteristics of CMML, unlike single Srsf2 or Tet2 mutation. This model represents a significant step toward building high fidelity and genetically tractable models of CMML.

The small PPR protein SPR2 interacts with PPR-SMR1 to facilitate the splicing of introns in maize mitochondria.

Splicing of plant mitochondrial introns is facilitated by numerous nucleus-encoded protein factors. Although some splicing factors have been identified in plants, the mechanism underlying mitochondrial intron splicing remains largely unclear. In this study, we identified a small P-type pentatricopeptide repeat (PPR) protein containing merely four PPR repeats, small PPR protein 2 (SPR2), which is required for the splicing of more than half of the introns in maize (Zea mays) mitochondria. Null mutations of Spr2 severely impair the splicing of 15 out of the 22 mitochondrial Group II introns, resulting in substantially decreased mature transcripts, which abolished the assembly and activity of mitochondrial complex I. Consequently, embryogenesis and endosperm development were arrested in the spr2 mutants. Yeast two-hybrid, luciferase complementation imaging, bimolecular fluorescence complementation, and semi-in vivo pull-down analyses indicated that SPR2 interacts with small MutS-related domain protein PPR-SMR1, both of which are required for the splicing of 13 introns. In addition, SPR2 and/or PPR-SMR1 interact with other splicing factors, including PPR proteins EMPTY PERICARP16, PPR14, and chloroplast RNA splicing and ribosome maturation (CRM) protein Zm-mCSF1, which participate in the splicing of specific intron(s) of the 13 introns. These results prompt us to propose that SPR2/PPR-SMR1 serves as the core component of a splicing complex and possibly exerts the splicing function through a dynamic interaction with specific substrate recognizing PPR proteins in mitochondria.

The Effects of Tail Truncations of Pre‐Messenger RNA Splicing Protein Dib1

The spliceosome is a multi‐megadalton ribonucleoprotein complex that catalyzes the removal of non‐coding introns and the ligation of coding exons from pre‐messenger RNA. This process is crucial in the transformation of pre‐messenger RNA into messenger RNA, a template that is readable by the ribosome and allows for the production of a functional protein. Significantly, the spliceosome is constructed anew on every pre‐mRNA. This construction entails over 140 protein factors that associate and dissociate throughout the splicing cycle, causing the conformational rearrangements and transesterification reactions necessary to yield the mature RNA template. Therefore, rather than a singular composition of proteins and RNAs being known as “the spliceosome,” this term actually refers to several different complexes which are distinct in their makeup and their role within the splicing cycle. Construction of a catalytic splicing complex occurs after a series of early assembly steps. Amongst the proteins involved in these early assembly steps is a 16.8 kDa protein, Dib1, that is present in the pre‐catalytic spliceosome but absent from the catalytically active conformation. The loss of Dib1 at this critical juncture hints at a potential role helping to facilitate the transition from pre‐catalytic to catalytic. Previous work on Dib1 has revealed that not only is it required for splicing, but the human ortholog, hDim1, has been shown to possess autocleavage activity in vitro where the last 14 amino acids are cleaved from its C‐terminal tail. Thus, we are interested in whether the C‐terminus impacts the function of the protein as it pertains to its role in splicing. In order to elucidate the importance of this region, we have constructed a series of C‐terminal truncations of varying lengths within Dib1 in order to assess any resulting splicing defects. These defects have been monitored through the use of growth assays and splicing assays to ascertain not only the overall importance of this region, but also the specific length required to ensure splicing abilities similar to wildtype Dib1 within the model organism S. cerevisiae. Overall, we observe that Dib1 can tolerate a large deletion before affecting cell growth and splicing.

Putting Humpty Dumpty Back Together Again: What Does Protein Quantification Mean in Bottom-Up Proteomics?

Bottom-up proteomics provides peptide measurements and has been invaluable for moving proteomics into large-scale analyses. Commonly, a single quantitative value is reported for each protein-coding gene by aggregating peptide quantities into protein groups following protein inference or parsimony. However, given the complexity of both RNA splicing and post-translational protein modification, it is overly simplistic to assume that all peptides that map to a singular protein-coding gene will demonstrate the same quantitative response. By assuming that all peptides from a protein-coding sequence are representative of the same protein, we may miss the discovery of important biological differences. To capture the contributions of existing proteoforms, we need to reconsider the practice of aggregating protein values to a single quantity per protein-coding gene.

Brain tissue transcriptome analysis for progressive supranuclear palsy disease.

Progressive supranuclear palsy (PSP) is an sporadic, uncommon neurological disorder. It is considered the second most frequent form of atypical parkinsonism. It is characterized by the presence of neurofibrillary tangles in the brainstem, basal ganglia, and cerebellum. Nowadays, there is no apparent trigger of this disease, many MAPT mutations related to the inclusion of exon 10 and environmental factors have been reported as significant risk factors, but much more remains to be elucidated. There exist a wide range of bioinformatics tools, and available microarray databases that could help us study dysregulated cellular and molecular processes leading usto find new therapeutic targets to test possible drugs METHOD: In this study, we seek online available transcriptomic profile data of confirmed PSP patients. We used ArrayExpress microarray dataset from medial temporal lobe (E-MEXP-2280). Raw data were processed and normalized in R system packages; differential expression analysis was performed with limma package, differentially expressed genes (DEGs) were selected with a adjusted p-value<0.05, and a logarithmic fold change greater than 0.5, and minor -0.5, and further functional enrichment analysis and KEGGp analysis were conducted with topGO and limma packages. After differential expression analysis we obtain a total of 640 differentially expressed genes, amount them, 382 were down regulated and 258 upregulated. We observed an increased up-regulated expression of genes related with developmental process, glutamatergic synapse, and transcription activity. And down-regulation of genes related to mRNA processing, endoplasmic reticulum, and extracellular exosome. Our results agree with previous reports, where elevated brain glutamate levels have been found in several brain sites in PSP; also endoplasmic reticulum dysregulated unfolded protein response has been related with PSP pathology. Furthermore, we observe a marked dysregulation of genes involved in mRNA processing, this agrees with previous reports were RNA splicing and RNA binding proteins could be playing an essential physiopathological role in the progression of some tauopathies. We propose that these DEGs could be useful as new biomarkers and possible therapeutic targets for PSP.

Developing biomarkers of TDP‐43‐related splicing dysfunction in frontotemporal dementia

AbstractBackgroundFrontotemporal dementia (FTD) is a rapidly progressive neurodegenerative disorder characterized by prominent atrophy of the frontal and temporal lobes and marked language and/or behavioral disturbances. Mislocalization of the TAR DNA‐binding protein 43 (TDP‐43) – characterized by both its nuclear loss and its cytosolic accumulation – occurs in up to 50% of FTD cases. Recent evidence from our group and others demonstrates that loss of TDP‐43‐dependent splicing activity occurs in FTD patient brains, is an early disease phenomenon, and contributes directly to neuronal death. Monitoring the products of TDP‐43 related mis‐splicing may, thus, yield promising biomarker candidates of early cellular dysfunction in FTD.MethodWe propose a series of proteogenomic studies in human iPSC neurons, post‐mortem brain, and patient biospecimens to identify and develop protein‐based biomarkers of RNA mis‐splicing in FTD. First, we will identify pathologic RNA splicing changes and protein products of mis‐splicing that occur in the context of TDP‐43 depletion in iPSC‐derived neurons. We will next determine which of these predicted transcripts and de novo proteins occur in post‐mortem human FTD brains, as a crucial first step in biomarker development. Finally, we will develop CSF biomarkers of FTD‐related mis‐splicing through a combination of targeted proteomics and ELISA‐based assays of lead candidate biomarkers.ResultUsing CRISPRi knockdown of TDP‐43 in i3Neurons followed by total RNAseq, we detected cryptic exon formation in STMN2 as well as hundreds of additional transcripts. We developed a proteogenomic informatic pipeline to detect de novo protein products of mis‐spliced transcripts in TDP‐43 deficient i3Neurons, revealing two in‐frame de novo peptides in the MYO18A transcript. Furthermore, we detected 38 of these mis‐spliced transcripts in ALS/FTD post‐mortem cortex, including MYO18A.ConclusionThese preliminary studies indicate that our i3Neuron CRISPRi pipeline can predict disease‐relevant markers of TDP‐43 mis‐splicing and suggest a proteogenomic workflow for development of novel protein‐based biomarkers of mis‐spliced transcripts.Together, these efforts will yield early biomarkers of TDP‐43 related cellular dysfunction that can act as surrogate measures of disease onset and progression in FTD.

Transcription Factors, R-Loops and Deubiquitinating Enzymes: Emerging Targets in Myelodysplastic Syndromes and Acute Myeloid Leukemia.

Simple SummaryThe advent of DNA massive sequencing technologies has allowed for the first time an extensive look into the heterogeneous spectrum of genes and mutations underpinning myelodysplastic syndromes (MDSs) and acute myeloid leukemia (AML). In this review, we wish to explore the most recent advances and the rationale for the potential therapeutic interest of three main actors in myelo-leukemic transformation: transcription factors that govern myeloid differentiation; RNA splicing factors, which ensure proper mRNA maturation and whose mutations increase R-loops formation; and deubiquitinating enzymes, which contribute to genome stability in hematopoietic stem cells (HSCs).Myeloid neoplasms encompass a very heterogeneous family of diseases characterized by the failure of the molecular mechanisms that ensure a balanced equilibrium between hematopoietic stem cells (HSCs) self-renewal and the proper production of differentiated cells. The origin of the driver mutations leading to preleukemia can be traced back to HSC/progenitor cells. Many properties typical to normal HSCs are exploited by leukemic stem cells (LSCs) to their advantage, leading to the emergence of a clonal population that can eventually progress to leukemia with variable latency and evolution. In fact, different subclones might in turn develop from the original malignant clone through accumulation of additional mutations, increasing their competitive fitness. This process ultimately leads to a complex cancer architecture where a mosaic of cellular clones—each carrying a unique set of mutations—coexists. The repertoire of genes whose mutations contribute to the progression toward leukemogenesis is broad. It encompasses genes involved in different cellular processes, including transcriptional regulation, epigenetics (DNA and histones modifications), DNA damage signaling and repair, chromosome segregation and replication (cohesin complex), RNA splicing, and signal transduction. Among these many players, transcription factors, RNA splicing proteins, and deubiquitinating enzymes are emerging as potential targets for therapeutic intervention.

Nanomechanical Phenotypes in Cardiac Myosin-Binding Protein C Mutants That Cause Hypertrophic Cardiomyopathy.

Hypertrophic cardiomyopathy (HCM) is a disease of the myocardium caused by mutations in sarcomeric proteins with mechanical roles, such as the molecular motor myosin. Around half of the HCM-causing genetic variants target contraction modulator cardiac myosin-binding protein C (cMyBP-C), although the underlying pathogenic mechanisms remain unclear since many of these mutations cause no alterations in protein structure and stability. As an alternative pathomechanism, here we have examined whether pathogenic mutations perturb the nanomechanics of cMyBP-C, which would compromise its modulatory mechanical tethers across sliding actomyosin filaments. Using single-molecule atomic force spectroscopy, we have quantified mechanical folding and unfolding transitions in cMyBP-C domains targeted by HCM mutations that do not induce RNA splicing alterations or protein thermodynamic destabilization. Our results show that domains containing mutation R495W are mechanically weaker than wild-type at forces below 40 pN and that R502Q mutant domains fold faster than wild-type. None of these alterations are found in control, nonpathogenic variants, suggesting that nanomechanical phenotypes induced by pathogenic cMyBP-C mutations contribute to HCM development. We propose that mutation-induced nanomechanical alterations may be common in mechanical proteins involved in human pathologies.

DNA methylation-mediated modulation of rapid desiccation tolerance acquisition and dehydration stress memory in the resurrection plant Boea hygrometrica.

Pre-exposure of plants to various abiotic conditions confers improved tolerance to subsequent stress. Mild drought acclimation induces acquired rapid desiccation tolerance (RDT) in the resurrection plant Boea hygrometrica, but the mechanisms underlying the priming and memory processes remain unclear. In this study, we demonstrated that drought acclimation-induced RDT can be maintained for at least four weeks but was completely erased after 18 weeks based on a combination of the phenotypic and physiological parameters. Global transcriptome analysis identified several RDT-specific rapid dehydration-responsive genes related to cytokinin and phospholipid biosynthesis, nitrogen and carbon metabolism, and epidermal morphogenesis, most of which were pre-induced by drought acclimation. Comparison of whole-genome DNA methylation revealed dehydration stress-responsive hypomethylation in the CG, CHG, and CHH contexts and acclimation-induced hypermethylation in the CHH context of the B. hygrometrica genome, consistent with the transcriptional changes in methylation pathway genes. As expected, the global promoter and gene body methylation levels were negatively correlated with gene expression levels in both acclimated and dehydrated plants but showed no association with transcriptional divergence during the procedure. Nevertheless, the promoter methylation variations in the CG and CHG contexts were significantly associated with the differential expression of genes required for fundamental genetic processes of DNA conformation, RNA splicing, translation, and post-translational protein modification during acclimation, growth, and rapid dehydration stress response. It was also associated with the dehydration stress-induced upregulation of memory genes, including pre-mRNA-splicing factor 38A, vacuolar amino acid transporter 1-like, and UDP-sugar pyrophosphorylase, which may contribute directly or indirectly to the improvement of dehydration tolerance in B. hygrometrica plants. Altogether, our findings demonstrate the potential implications of DNA methylation in dehydration stress memory and, therefore, provide a molecular basis for enhanced dehydration tolerance in plants induced by drought acclimation.

Mg2+ Transporters in Digestive Cancers.

Despite magnesium (Mg2+) representing the second most abundant cation in the cell, its role in cellular physiology and pathology is far from being elucidated. Mg2+ homeostasis is regulated by Mg2+ transporters including Mitochondrial RNA Splicing Protein 2 (MRS2), Transient Receptor Potential Cation Channel Subfamily M, Member 6/7 (TRPM6/7), Magnesium Transporter 1 (MAGT1), Solute Carrier Family 41 Member 1 (SCL41A1), and Cyclin and CBS Domain Divalent Metal Cation Transport Mediator (CNNM) proteins. Recent data show that Mg2+ transporters may regulate several cancer cell hallmarks. In this review, we describe the expression of Mg2+ transporters in digestive cancers, the most common and deadliest malignancies worldwide. Moreover, Mg2+ transporters’ expression, correlation and impact on patient overall and disease-free survival is analyzed using Genotype Tissue Expression (GTEx) and The Cancer Genome Atlas (TCGA) datasets. Finally, we discuss the role of these Mg2+ transporters in the regulation of cancer cell fates and oncogenic signaling pathways.