Variant-specific effects define the phenotypic spectrum of HNRNPH2-associated neurodevelopmental disorders in males

Bain type of X-linked syndromic intellectual developmental disorder, caused by pathogenic missense variants in HRNRPH2, was initially described in six female individuals affected by moderate-to-severe neurodevelopmental delay. Although it was initially postulated that the condition would not be compatible with life in males, several affected male individuals harboring pathogenic variants in HNRNPH2 have since been documented. However, functional in-vitro analyses of identified variants have not been performed and, therefore, possible genotype–phenotype correlations remain elusive. Here, we present eight male individuals, including a pair of monozygotic twins, harboring pathogenic or likely pathogenic HNRNPH2 variants. Notably, we present the first individuals harboring nonsense or frameshift variants who, similarly to an individual harboring a de novo p.(Arg29Cys) variant within the first quasi-RNA-recognition motif (qRRM), displayed mild developmental delay, and developed mostly autistic features and/or psychiatric co-morbidities. Additionally, we present two individuals harboring a recurrent de novo p.(Arg114Trp), within the second qRRM, who had a severe neurodevelopmental delay with seizures. Functional characterization of the three most common HNRNPH2 missense variants revealed dysfunctional nucleocytoplasmic shuttling of proteins harboring the p.(Arg206Gln) and p.(Pro209Leu) variants, located within the nuclear localization signal, whereas proteins with p.(Arg114Trp) showed reduced interaction with members of the large assembly of splicing regulators (LASR). Moreover, RNA-sequencing of primary fibroblasts of the individual harboring the p.(Arg114Trp) revealed substantial alterations in the regulation of alternative splicing along with global transcriptome changes. Thus, we further expand the clinical and variant spectrum in HNRNPH2-associated disease in males and provide novel molecular insights suggesting the disorder to be a spliceopathy on the molecular level.

hnRNPH1 recruits PTBP2 and SRSF3 to cooperatively modulate alternative pre-mRNA splicing in germ cells and is essential for spermatogenesis and oogenesis

Coordinated regulation of alternative pre-mRNA splicing is essential for germ cell development. However, the molecular mechanism underlying that control alternative mRNA expression during germ cell development remains poorly understood. Herein, we showed that hnRNPH1, an RNA-binding protein, is highly expressed in the reproductive system and localized in the chromosomes of meiotic cells but excluded from the XY body in pachytene spermatocytes and recruits the splicing regulators PTBP2 and SRSF3 and cooperatively regulates the alternative splicing of the critical genes that are required for spermatogenesis. Conditional knockout Hnrnph1 in spermatogenic cells caused many abnormal splicing events that affect genes related to meiosis and communication between germ cells and Sertoli cells, characterized by asynapsis of chromosomes and impairment of germ-Sertoli communications, ultimately leading to male sterility. We further showed that hnRNPH1 could directly bind to SPO11 and recruit the splicing regulators PTBP2 and SRSF3 to regulate the alternative splicing of the target genes cooperatively. Strikingly, Hnrnph1 germline-specific mutant female mice were also infertile, and Hnrnph1-deficient oocytes exhibited a similar defective synapsis and cell-cell junction as shown in Hnrnph1-deficient male germ cells. Collectively, our data reveal an essential role for hnRNPH1 in regulating pre-mRNA splicing during spermatogenesis and oogenesis and support a molecular model whereby hnRNPH1 governs a network of alternative splicing events in germ cells via recruiting PTBP2 and SRSF3.

Structural basis of the interaction between SETD2 methyltransferase and hnRNP L paralogs for governing co-transcriptional splicing

The RNA recognition motif (RRM) binds to nucleic acids as well as proteins. More than one such domain is found in the pre-mRNA processing hnRNP proteins. While the mode of RNA recognition by RRMs is known, the molecular basis of their protein interaction remains obscure. Here we describe the mode of interaction between hnRNP L and LL with the methyltransferase SETD2. We demonstrate that for the interaction to occur, a leucine pair within a highly conserved stretch of SETD2 insert their side chains in hydrophobic pockets formed by hnRNP L RRM2. Notably, the structure also highlights that RRM2 can form a ternary complex with SETD2 and RNA. Remarkably, mutating the leucine pair in SETD2 also results in its reduced interaction with other hnRNPs. Importantly, the similarity that the mode of SETD2-hnRNP L interaction shares with other related protein-protein interactions reveals a conserved design by which splicing regulators interact with one another.

Role of Alternative Splicing in Sex Determination in Vertebrates

During the process of sex determination, a germ-cell-containing undifferentiated gonad is converted into either a male or a female reproductive organ. Both the composition of sex chromosomes and the environment determine sex in vertebrates. It is assumed that transcription level regulation drives this cascade of mechanisms; however, transcription factors can alter gene expression beyond transcription initiation by controlling pre-mRNA splicing and thereby mRNA isoform production. Using the key time window in sex determination and gonad development in mice, it has been reported that new non-transcriptional events, such as alternative splicing, could play a key role in sex determination in mammals. We know the role of key regulatory factors, like WT1(+/–KTS) or FGFR2(b/c) in pre-mRNA splicing and sex determination, indicating that important steps in the vertebrate sex determination process probably operate at a post-transcriptional level. Here, we discuss the role of pre-mRNA splicing regulators in sex determination in vertebrates, focusing on the new RNA-seq data reported from mice fetal gonadal transcriptome.

Dysregulated coordination of MAPT exon 2 and exon 10 splicing underlies different tau pathologies in PSP and AD

ABSTRACTUnderstanding regulation of MAPT splicing is important to the etiology of many nerurodegenerative diseases, including Alzheimer disease (AD) and progressive supranuclear palsy (PSP), in which different tau isoforms accumulate in pathologic inclusions. MAPT, the gene encoding the tau protein, undergoes complex alternative pre-mRNA splicing to generate six isoforms. Tauopathies can be categorized by the presence of tau aggregates containing either 3 (3R) or 4 (4R) microtubule binding domain repeats (determined by inclusion/exclusion of exon 10), but the role of the N terminal domain of the protein, determined by inclusion/exclusion of exons 2 and 3 has been less well studied. Using an unbiased correlational screen in human brain tissue, we observed coordination of MAPT exons 2 and 10 splicing. Expression of exon 2 splicing regulators and subsequently exon 2 inclusion are differentially disrupted in PSP and AD brain, resulting in the accumulation of 1N4R isoforms in PSP and 0N isoforms in AD temporal cortex. Furthermore, we identified different N-terminal isoforms of tau present in neurofibrillary tangles, dystrophic neurites and tufted astrocytes, indicating a role for differential N-terminal splicing in the development of disparate tau neuropathologies. We conclude that N-terminal splicing and combinatorial regulation with exon 10 inclusion/exclusion is likely to be important to our understanding of tauopathies.

Loss of Muscleblind Splicing Factor Shortens C. elegans Lifespan by Reducing the Activity of p38 MAPK/PMK-1 and Transcription Factors ATF-7 and Nrf/SKN-1

Muscleblind-like splicing regulators (MBNLs) are RNA-binding factors that have an important role in developmental processes. Dysfunction of these factors is a key contributor of different neuromuscular degenerative disorders, including Myotonic Dystrophy type 1 (DM1). Since DM1 is a multisystemic disease characterized by symptoms resembling accelerated aging, we asked which cellular processes do MBNLs regulate that make them necessary for normal lifespan. By utilizing the model organism Caenorhabditis elegans, we found that loss of MBL-1 (the sole ortholog of mammalian MBNLs), which is known to be required for normal lifespan, shortens lifespan by decreasing the activity of p38 MAPK/PMK-1 as well as the function of transcription factors ATF-7 and SKN-1. Furthermore, we show that mitochondrial stress caused by knockdown of mitochondrial electron transport chain components promotes the longevity of mbl-1 mutants in a partially PMK-1-dependent manner. Together, the data establish a mechanism of how DM1-associated loss of muscleblind affects lifespan. Furthermore, this study suggests that mitochondrial stress could alleviate symptoms caused by the dysfunction of muscleblind splicing factor, creating a potential approach to investigate for therapy.

Mechanistic Insights of Aberrant Splicing with Splicing Factor Mutations Found in Myelodysplastic Syndromes

Pre-mRNA splicing is an essential process for gene expression in higher eukaryotes, which requires a high order of accuracy. Mutations in splicing factors or regulatory elements in pre-mRNAs often result in many human diseases. Myelodysplastic syndrome (MDS) is a heterogeneous group of chronic myeloid neoplasms characterized by many symptoms and a high risk of progression to acute myeloid leukemia. Recent findings indicate that mutations in splicing factors represent a novel class of driver mutations in human cancers and affect about 50% of Myelodysplastic syndrome (MDS) patients. Somatic mutations in MDS patients are frequently found in genes SF3B1, SRSF2, U2AF1, and ZRSR2. Interestingly, they are involved in the recognition of 3′ splice sites and exons. It has been reported that mutations in these splicing regulators result in aberrant splicing of many genes. In this review article, we first describe molecular mechanism of pre-mRNA splicing as an introduction and mainly focus on those four splicing factors to describe their mutations and their associated aberrant splicing patterns.

Alternative Splicing Regulates the Physiological Adaptation of the Mouse Hind Limb Postural and Phasic Muscles to Microgravity

Muscle atrophy and fiber type alterations are well-characterized physiological adaptations to microgravity with both understood to be primarily regulated by differential gene expression (DGE). While microgravity-induced DGE has been extensively investigated, adaptations to microgravity due to alternative splicing (AS) have not been studied in a mammalian model. We sought to comprehensively elucidate the transcriptomic underpinnings of microgravity-induced muscle phenotypes in mice by evaluating both DGE and changes in AS due to extended spaceflight. Tissue sections and total RNA were isolated from the gastrocnemius and quadriceps, postural and phasic muscles of the hind limb, respectively, of 32-week-old female BALB/c mice exposed to microgravity or ground control conditions for nine weeks. Immunohistochemistry disclosed muscle type-specific physiological adaptations to microgravity that included i) a pronounced reduction in muscle fiber cross-sectional area in both muscles and ii) a prominent slow-to-fast fiber type transition in the gastrocnemius. RNA sequencing revealed that DGE and AS varied across postural and phasic muscle types with preferential employment of DGE in the gastrocnemius and AS in the quadriceps. Gene ontology analysis indicated that DGE and AS regulate distinct molecular processes. Various non-differentially expressed transcripts encoding musculoskeletal proteins (Tnnt3, Tnnt1, Neb, Ryr1, and Ttn) and muscle-specific RNA binding splicing regulators (Mbnl1 and Rbfox1) were found to have significant changes in AS that altered critical functional domains of their protein products. In striking contrast, microgravity-induced differentially expressed genes were associated with lipid metabolism and mitochondrial function. Our work serves as the first comprehensive investigation of coordinate changes in DGE and AS in large limb muscles across spaceflight. We propose that substantial remodeling of pre-mRNA by AS is a major component of transcriptomic adaptation of skeletal muscle to microgravity. The alternatively spliced genes identified here could be targeted by small molecule splicing regulator therapies to address microgravity-induced changes in muscle during spaceflight.

Structural basis of the interaction between SETD2 methyltransferase and hnRNP L paralogs for governing co-transcriptional splicing

The RNA recognition motif (RRM) binds to nucleic acids as well as proteins. More than one such domain is found in the pre-mRNA processing hnRNP proteins. While the mode of RNA recognition by RRMs is known, the molecular basis of their protein interaction remains obscure. Here we describe the mode of interaction between hnRNP L and LL with the methyltransferase SETD2. We demonstrate that for the interaction to occur, a leucine pair within a highly conserved stretch of SETD2 insert their side chains in hydrophobic pockets formed by hnRNP L RRM2. Notably, the structure also highlights that RRM2 can form a ternary complex with SETD2 and RNA. Remarkably, mutating the leucine pair in SETD2 also results in its reduced interaction with other hnRNPs. Importantly, the similarity that the mode of SETD2-hnRNP L interaction shares with other related protein-protein interactions reveals a conserved design by which splicing regulators interact with one another.

Functional characterization of splicing regulatory elements

Background: RNA binding protein-RNA interactions mediate a variety of processes including pre-mRNA splicing, translation, decay, polyadenylation and many others. Previous high-throughput studies have characterized general sequence features associated with increased and decreased splicing of certain exons, but these studies are limited by not knowing the mechanisms, and in particular, the mediating RNA binding proteins, underlying these associations. Results: Here we utilize ENCODE data from diverse data modalities to identify functional splicing regulatory elements and their associated RNA binding proteins. We identify features which make splicing events more sensitive to depletion of RNA binding proteins, as well as which RNA binding proteins act as splicing regulators sensitive to depletion. To analyze the sequence determinants underlying RBP-RNA interactions impacting splicing, we assay tens of thousands of sequence variants in a high-throughput splicing reporter called Vex-seq and confirm a small subset in their endogenous loci using CRISPR base editors. Finally, we leverage other large transcriptomic datasets to confirm the importance of RNA binding proteins which we designed experiments around and identify additional RBPs which may act as additional splicing regulators of the exons studied. Conclusions: This study identifies sequence and other features underlying splicing regulation mediated specific RNA binding proteins, as well as validates and identifies other potentially important regulators of splicing in other large transcriptomic datasets.