PRPF8-mediated dysregulation of hBrr2 helicase disrupts human spliceosome kinetics and 5´-splice-site selection causing tissue-specific defects.

The carboxy-terminus of the spliceosomal protein PRPF8, which regulates the RNA helicase Brr2, is a hotspot for mutations causing retinitis pigmentosa-type 13, with unclear role in human splicing and tissue-specificity mechanism. We used patient induced pluripotent stem cells-derived cells, carrying the heterozygous PRPF8 c.6926 A > C (p.H2309P) mutation to demonstrate retinal-specific endophenotypes comprising photoreceptor loss, apical-basal polarity and ciliary defects. Comprehensive molecular, transcriptomic, and proteomic analyses revealed a role of the PRPF8/Brr2 regulation in 5'-splice site (5'SS) selection by spliceosomes, for which disruption impaired alternative splicing and weak/suboptimal 5'SS selection, and enhanced cryptic splicing, predominantly in ciliary and retinal-specific transcripts. Altered splicing efficiency, nuclear speckles organisation, and PRPF8 interaction with U6 snRNA, caused accumulation of active spliceosomes and poly(A)+ mRNAs in unique splicing clusters located at the nuclear periphery of photoreceptors. Collectively these elucidate the role of PRPF8/Brr2 regulatory mechanisms in splicing and the molecular basis of retinal disease, informing therapeutic approaches.

ST3 beta-galactoside alpha-2,3-sialyltransferase 1 (ST3Gal1) synthesis of Siglec ligands mediates anti-tumour immunity in prostate cancer.

Immune checkpoint blockade has yet to produce robust anti-cancer responses for prostate cancer. Sialyltransferases have been shown across several solid tumours, including breast, melanoma, colorectal and prostate to promote immune suppression by synthesising sialoglycans, which act as ligands for Siglec receptors. We report that ST3 beta-galactoside alpha-2,3-sialyltransferase 1 (ST3Gal1) levels negatively correlate with androgen signalling in prostate tumours. We demonstrate that ST3Gal1 plays an important role in modulating tumour immune evasion through the synthesises of sialoglycans with the capacity to engage the Siglec-7 and Siglec-9 immunoreceptors preventing immune clearance of cancer cells. Here, we provide evidence of the expression of Siglec-7/9 ligands and their respective immunoreceptors in prostate tumours. These interactions can be modulated by enzalutamide and may maintain immune suppression in enzalutamide treated tumours. We conclude that the activity of ST3Gal1 is critical to prostate cancer anti-tumour immunity and provide rationale for the use of glyco-immune checkpoint targeting therapies in advanced prostate cancer.

Differential expression of paralog RNA binding proteins establishes a dynamic splicing program required for normal cerebral cortex development.

Sam68 and SLM2 are paralog RNA binding proteins (RBPs) expressed in the cerebral cortex and display similar splicing activities. However, their relative functions during cortical development are unknown. We found that these RBPs exhibit an opposite expression pattern during development. Sam68 expression declines postnatally while SLM2 increases after birth, and this developmental pattern is reinforced by hierarchical control of Sam68 expression by SLM2. Analysis of Sam68:Slm2 double knockout (Sam68:Slm2dko) mice revealed hundreds of exons that respond to joint depletion of these proteins. Moreover, parallel analysis of single and double knockout cortices indicated that exons regulated mainly by SLM2 are characterized by a dynamic splicing pattern during development, whereas Sam68-dependent exons are spliced at relatively constant rates. Dynamic splicing of SLM2-sensitive exons is completely suppressed in the Sam68:Slm2dko developing cortex. Sam68:Slm2dko mice die perinatally with defects in neurogenesis and in neuronal differentiation, and develop a hydrocephalus, consistent with splicing alterations in genes related to these biological processes. Thus, our study reveals that developmental control of separate Sam68 and Slm2 paralog genes encoding homologous RBPs enables the orchestration of a dynamic splicing program needed for brain development and viability, while ensuring a robust redundant mechanism that supports proper cortical development.

The role of GCNT1 mediated O-glycosylation in aggressive prostate cancer.

Prostate cancer is the most common cancer in men and a major cause of cancer related deaths worldwide. Nearly all affected men develop resistance to current therapies and there is an urgent need to develop new treatments for advanced disease. Aberrant glycosylation is a common feature of cancer cells implicated in all of the hallmarks of cancer. A major driver of aberrant glycosylation in cancer is the altered expression of glycosylation enzymes. Here, we show that GCNT1, an enzyme that plays an essential role in the formation of core 2 branched O-glycans and is crucial to the final definition of O-glycan structure, is upregulated in aggressive prostate cancer. Using in vitro and in vivo models, we show GCNT1 promotes the growth of prostate tumours and can modify the glycome of prostate cancer cells, including upregulation of core 2 O-glycans and modifying the O-glycosylation of secreted glycoproteins. Furthermore, using RNA sequencing, we find upregulation of GCNT1 in prostate cancer cells can alter oncogenic gene expression pathways important in tumour growth and metastasis. Our study highlights the important role of aberrant O-glycosylation in prostate cancer progression and provides novel insights regarding the mechanisms involved.

Epithelial specific splicing regulator proteins as emerging oncogenes in aggressive prostate cancer.

Prostate cancer progression is connected to the activity of conventional oncogenes and tumour suppressors and driven by circulating steroid hormones. A key issue has been how to identify and care for aggressively developing prostate tumours. Here we discuss how expression of the splicing regulators ESRP1 and ESRP2, and how their role as "masterminds" of epithelial splicing patterns, have been identified as markers of aggressively proliferating prostate primary tumours. We suggest that the origin of prostate cancer within epithelial cells, and the subsequent association of ESRP1 and ESRP2 expression with more aggressive disease progression, identify ESRP1 and ESRP2 as lineage survival oncogenes. To move this field on in the future it will be important to identify the gene expression targets controlled by ESRP1/2 that regulate prostate cancer proliferation. Potential future therapies could be designed to target ESRP1 and ESRP2 protein activity or their regulated splice isoforms in aggressive prostate tumours. Design of these therapies is potentially complicated by the risk of producing a more mesenchymal splicing environment that might promote tumour metastasis.

ST6GAL1-mediated aberrant sialylation promotes prostate cancer progression.

Aberrant glycosylation is a universal feature of cancer cells, and cancer-associated glycans have been detected in virtually every cancer type. A common change in tumour cell glycosylation is an increase in α2,6 sialylation of N-glycans, a modification driven by the sialyltransferase ST6GAL1. ST6GAL1 is overexpressed in numerous cancer types, and sialylated glycans are fundamental for tumour growth, metastasis, immune evasion, and drug resistance, but the role of ST6GAL1 in prostate cancer is poorly understood. Here, we analyse matched cancer and normal tissue samples from 200 patients and verify that ST6GAL1 is upregulated in prostate cancer tissue. Using MALDI imaging mass spectrometry (MALDI-IMS), we identify larger branched α2,6 sialylated N-glycans that show specificity to prostate tumour tissue. We also monitored ST6GAL1 in plasma samples from >400 patients and reveal ST6GAL1 levels are significantly increased in the blood of men with prostate cancer. Using both in vitro and in vivo studies, we demonstrate that ST6GAL1 promotes prostate tumour growth and invasion. Our findings show ST6GAL1 introduces α2,6 sialylated N-glycans on prostate cancer cells and raise the possibility that prostate cancer cells can secrete active ST6GAL1 enzyme capable of remodelling glycans on the surface of other cells. Furthermore, we find α2,6 sialylated N-glycans expressed by prostate cancer cells can be targeted using the sialyltransferase inhibitor P-3FAX -Neu5Ac. Our study identifies an important role for ST6GAL1 and α2,6 sialylated N-glycans in prostate cancer progression and highlights the opportunity to inhibit abnormal sialylation for the development of new prostate cancer therapeutics. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

YBX1-interacting small RNAs and RUNX2 can be blocked in primary bone cancer using CADD522.

Primary bone cancer (PBC) comprises several subtypes each underpinned by distinctive genetic drivers. This driver diversity produces novel morphological features and clinical behaviour that serendipitously makes PBC an excellent metastasis model. Here, we report that some transfer RNA-derived small RNAs termed tRNA fragments (tRFs) perform as a constitutive tumour suppressor mechanism by blunting a potential pro-metastatic protein-RNA interaction. This mechanism is reduced in PBC progression with a gradual loss of tRNAGlyTCC cleavage into 5' end tRF-GlyTCC when comparing low-grade, intermediate-grade and high-grade patient tumours. We detected recurrent activation of miR-140 leading to upregulated RUNX2 expression in high-grade patient tumours. Both tRF-GlyTCC and RUNX2 share a sequence motif in their 3' ends that matches the YBX1 recognition site known to stabilise pro-metastatic mRNAs. Investigating some aspects of this interaction network, gain- and loss-of-function experiments using small RNA mimics and antisense LNAs, respectively, showed that ectopic tRF-GlyTCC reduced RUNX2 expression and dispersed 3D micromass architecture in vitro. iCLIP sequencing revealed YBX1 physical binding to the 3' UTR of RUNX2. The interaction between YBX1, tRF-GlyTCC and RUNX2 led to the development of the RUNX2 inhibitor CADD522 as a PBC treatment. CADD522 assessment in vitro revealed significant effects on PBC cell behaviour. In xenograft mouse models, CADD522 as a single agent without surgery significantly reduced tumour volume, increased overall and metastasis-free survival and reduced cancer-induced bone disease. Our results provide insight into PBC molecular abnormalities that have led to the identification of new targets and a new therapeutic.

Clustered variants in the 5' coding region of TRA2B cause a distinctive neurodevelopmental syndrome.

PURPOSE: Transformer2 proteins (Tra2α and Tra2ß) control splicing patterns in human cells, and no human phenotypes have been associated with germline variants in these genes. The aim of this work was to associate germline variants in the TRA2B gene to a novel neurodevelopmental disorder. METHODS: A total of 12 individuals from 11 unrelated families who harbored predicted loss-of-function monoallelic variants, mostly de novo, were recruited. RNA sequencing and western blot analyses of Tra2ß-1 and Tra2ß-3 isoforms from patient-derived cells were performed. Tra2ß1-GFP, Tra2ß3-GFP and CHEK1 exon 3 plasmids were transfected into HEK-293 cells. RESULTS: All variants clustered in the 5' part of TRA2B, upstream of an alternative translation start site responsible for the expression of the noncanonical Tra2ß-3 isoform. All affected individuals presented intellectual disability and/or developmental delay, frequently associated with infantile spasms, microcephaly, brain anomalies, autism spectrum disorder, feeding difficulties, and short stature. Experimental studies showed that these variants decreased the expression of the canonical Tra2ß-1 isoform, whereas they increased the expression of the Tra2ß-3 isoform, which is shorter and lacks the N-terminal RS1 domain. Increased expression of Tra2ß-3-GFP were shown to interfere with the incorporation of CHEK1 exon 3 into its mature transcript, normally incorporated by Tra2ß-1. CONCLUSION: Predicted loss-of-function variants clustered in the 5' portion of TRA2B cause a new neurodevelopmental syndrome through an apparently dominant negative disease mechanism involving the use of an alternative translation start site and the overexpression of a shorter, repressive Tra2ß protein.

Pro-Survival Factor EDEM3 Confers Therapy Resistance in Prostate Cancer.

Prostate cancer is the most common cancer in men, and it is primarily driven by androgen steroid hormones. The glycosylation enzyme EDEM3 is controlled by androgen signalling and is important for prostate cancer viability. EDEM3 is a mannosidase that trims mannose from mis-folded glycoproteins, tagging them for degradation through endoplasmic reticulum-associated degradation. Here, we find that EDEM3 is upregulated in prostate cancer, and this is linked to poorer disease-free survival. Depletion of EDEM3 from prostate cancer cells induces an ER stress transcriptomic signature, and EDEM3 overexpression is cyto-protective against ER stressors. EDEM3 expression also positively correlates with genes involved in the unfolded protein response in prostate cancer patients, and its expression can be induced through exposure to radiation. Importantly, the overexpression of EDEM3 promotes radio-resistance in prostate cancer cells and radio-resistance can be reduced through depletion of EDEM3. Our data thus implicate increased levels of EDEM3 with a role in prostate cancer pathology and reveal a new therapeutic opportunity to sensitise prostate tumours to radiotherapy.

Cryptic splicing: common pathological mechanisms involved in male infertility and neuronal diseases.

High levels of transcription and alternative splicing are recognized hallmarks of gene expression in the testis and largely driven by cells in meiosis. Because of this, the male meiosis stage of the cell cycle is often viewed as having a relatively permissive environment for gene expression. In this review, we highlight recent findings that identify the RNA binding protein RBMXL2 as essential for male meiosis. RBMXL2 functions as a "guardian of the transcriptome" that protects against the use of aberrant (or "cryptic") splice sites that would disrupt gene expression. This newly discovered protective role during meiosis links with a wider field investigating mechanisms of cryptic splicing control that protect neurons from amyotrophic lateral sclerosis and Alzheimer's disease. We discuss how the mechanism repressing cryptic splicing patterns during meiosis evolved, and why it may be essential for sperm production and male fertility.

FUT8 Alpha-(1,6)-Fucosyltransferase in Cancer.

Aberrant glycosylation is a universal feature of cancer cells that can impact all steps in tumour progression from malignant transformation to metastasis and immune evasion. One key change in tumour glycosylation is altered core fucosylation. Core fucosylation is driven by fucosyltransferase 8 (FUT8), which catalyses the addition of α1,6-fucose to the innermost GlcNAc residue of N-glycans. FUT8 is frequently upregulated in cancer, and plays a critical role in immune evasion, antibody-dependent cellular cytotoxicity (ADCC), and the regulation of TGF-ß, EGF, α3ß1 integrin and E-Cadherin. Here, we summarise the role of FUT8 in various cancers (including lung, liver, colorectal, ovarian, prostate, breast, melanoma, thyroid, and pancreatic), discuss the potential mechanisms involved, and outline opportunities to exploit FUT8 as a critical factor in cancer therapeutics in the future.

Direct glia-to-neuron transdifferentiation gives rise to a pair of male-specific neurons that ensure nimble male mating.

Sexually dimorphic behaviours require underlying differences in the nervous system between males and females. The extent to which nervous systems are sexually dimorphic and the cellular and molecular mechanisms that regulate these differences are only beginning to be understood. We reveal here a novel mechanism by which male-specific neurons are generated in Caenorhabditis elegans through the direct transdifferentiation of sex-shared glial cells. This glia-to-neuron cell fate switch occurs during male sexual maturation under the cell-autonomous control of the sex-determination pathway. We show that the neurons generated are cholinergic, peptidergic, and ciliated putative proprioceptors which integrate into male-specific circuits for copulation. These neurons ensure coordinated backward movement along the mate's body during mating. One step of the mating sequence regulated by these neurons is an alternative readjustment movement performed when intromission becomes difficult to achieve. Our findings reveal programmed transdifferentiation as a developmental mechanism underlying flexibility in innate behaviour.

Tumour associated glycans: A route to boost immunotherapy?

While the development of immunotherapies for cancer treatment offer significant promise across several cancers, still only a small subset of patients respond to immune based monotherapies. As such, attention has turned to the development of combination therapies. These use conventional cancer treatments such as chemotherapy to sensitise tumours to immunotherapy. Here, we summarise key research, highlighting the exciting potential of tumour associated glycans as therapeutic targets to sensitise tumours to immunotherapy. When cells undergo carcinogenesis they reprogram their glyco-code. Several cancer associated glycans have been identified, and therapies targeting them are under development. Proteins containing carbohydrate binding domains (lectins) are expressed by many immune cell subtypes, and upon glycan binding, transduce immune modulatory signals that regulate the tumour immune microenvironment.

Androgen-regulated transcription of ESRP2 drives alternative splicing patterns in prostate cancer.

Prostate is the most frequent cancer in men. Prostate cancer progression is driven by androgen steroid hormones, and delayed by androgen deprivation therapy (ADT). Androgens control transcription by stimulating androgen receptor (AR) activity, yet also control pre-mRNA splicing through less clear mechanisms. Here we find androgens regulate splicing through AR-mediated transcriptional control of the epithelial-specific splicing regulator ESRP2. Both ESRP2 and its close paralog ESRP1 are highly expressed in primary prostate cancer. Androgen stimulation induces splicing switches in many endogenous ESRP2-controlled mRNA isoforms, including splicing switches correlating with disease progression. ESRP2 expression in clinical prostate cancer is repressed by ADT, which may thus inadvertently dampen epithelial splice programmes. Supporting this, treatment with the AR antagonist bicalutamide (Casodex) induced mesenchymal splicing patterns of genes including FLNB and CTNND1. Our data reveals a new mechanism of splicing control in prostate cancer with important implications for disease progression.

Alternative splicing in lung cancer.

Lung cancer has the highest mortality rate of all cancers worldwide. Lung cancer is a very heterogeneous disease that is often diagnosed at later stages which have a poor prognosis. Aberrant alternative splicing patterns found in lung cancer contribute to important cell functions. These include changes in splicing for the BCL2L1, MDM2, MDM4, NUMB and MET genes during lung tumourigenesis, to affect pathways involved in apoptosis, cell proliferation and cellular cohesion. Global analyses of RNASeq datasets suggest there may be many more potentially influential aberrant splicing events that need to be investigated in lung cancer. Changes in expression of the splicing factors that regulate alternative splicing events have also been identified in lung cancer. Of these, changes in expression of QKI, RBM4, RBM5, RBM6, RBM10 and SRSF1 proteins regulate many of the most frequently referenced aberrant splicing events in lung cancer. The expanding list of genes known to be aberrantly spliced in lung cancer along with the altered expression of splicing factors that regulate them are providing new clues as to how lung cancer develops, and how these events can be exploited for better treatment. This article is part of a Special Issue entitled: RNA structure and splicing regulation edited by Francisco Baralle, Ravindra Singh and Stefan Stamm.

An integrated transcriptional analysis of the developing human retina.

The scarcity of embryonic/foetal material as a resource for direct study means that there is still limited understanding of human retina development. Here, we present an integrated transcriptome analysis combined with immunohistochemistry in human eye and retinal samples from 4 to 19 post-conception weeks. This analysis reveals three developmental windows with specific gene expression patterns that informed the sequential emergence of retinal cell types and enabled identification of stage-specific cellular and biological processes, and transcriptional regulators. Each stage is characterised by a specific set of alternatively spliced transcripts that code for proteins involved in the formation of the photoreceptor connecting cilium, pre-mRNA splicing and epigenetic modifiers. Importantly, our data show that the transition from foetal to adult retina is characterised by a large increase in the percentage of mutually exclusive exons that code for proteins involved in photoreceptor maintenance. The circular RNA population is also defined and shown to increase during retinal development. Collectively, these data increase our understanding of human retinal development and the pre-mRNA splicing process, and help to identify new candidate disease genes.

An ancient germ cell-specific RNA-binding protein protects the germline from cryptic splice site poisoning.

Male germ cells of all placental mammals express an ancient nuclear RNA binding protein of unknown function called RBMXL2. Here we find that deletion of the retrogene encoding RBMXL2 blocks spermatogenesis. Transcriptome analyses of age-matched deletion mice show that RBMXL2 controls splicing patterns during meiosis. In particular, RBMXL2 represses the selection of aberrant splice sites and the insertion of cryptic and premature terminal exons. Our data suggest a Rbmxl2 retrogene has been conserved across mammals as part of a splicing control mechanism that is fundamentally important to germ cell biology. We propose that this mechanism is essential to meiosis because it buffers the high ambient concentrations of splicing activators, thereby preventing poisoning of key transcripts and disruption to gene expression by aberrant splice site selection.

RBMX family proteins connect the fields of nuclear RNA processing, disease and sex chromosome biology.

RBMX is a ubiquitously expressed nuclear RNA binding protein that is encoded by a gene on the X chromosome. RBMX belongs to a small protein family with additional members encoded by paralogs on the mammalian Y chromosome and other chromosomes. These RNA binding proteins are important for normal development, and also implicated in cancer and viral infection. At the molecular level RBMX family proteins contribute to splicing control, transcription and genome integrity. Establishing what endogenous genes and pathways are controlled by RBMX and its paralogs will have important implications for understanding chromosome biology, DNA repair and mammalian development. Here we review what is known about this family of RNA binding proteins, and identify important current questions about their functions.

Androgen-dependent alternative mRNA isoform expression in prostate cancer cells.

Background: Androgen steroid hormones are key drivers of prostate cancer. Previous work has shown that androgens can drive the expression of alternative mRNA isoforms as well as transcriptional changes in prostate cancer cells. Yet to what extent androgens control alternative mRNA isoforms and how these are expressed and differentially regulated in prostate tumours is unknown. Methods: Here we have used RNA-Seq data to globally identify alternative mRNA isoform expression under androgen control in prostate cancer cells, and profiled the expression of these mRNA isoforms in clinical tissue. Results: Our data indicate androgens primarily switch mRNA isoforms through alternative promoter selection. We detected 73 androgen regulated alternative transcription events, including utilisation of 56 androgen-dependent alternative promoters, 13 androgen-regulated alternative splicing events, and selection of 4 androgen-regulated alternative 3' mRNA ends. 64 of these events are novel to this study, and 26 involve previously unannotated isoforms. We validated androgen dependent regulation of 17 alternative isoforms by quantitative PCR in an independent sample set. Some of the identified mRNA isoforms are in genes already implicated in prostate cancer (including LIG4, FDFT1 and RELAXIN), or in genes important in other cancers (e.g. NUP93 and MAT2A). Importantly, analysis of transcriptome data from 497 tumour samples in the TGCA prostate adenocarcinoma (PRAD) cohort identified 13 mRNA isoforms (including TPD52, TACC2 and NDUFV3) that are differentially regulated in localised prostate cancer relative to normal tissue, and 3 ( OSBPL1A, CLK3 and TSC22D3) which change significantly with Gleason grade and  tumour stage. Conclusions: Our findings dramatically increase the number of known androgen regulated isoforms in prostate cancer, and indicate a highly complex response to androgens in prostate cancer cells that could be clinically important.

Disrupted alternative splicing for genes implicated in splicing and ciliogenesis causes PRPF31 retinitis pigmentosa.

Mutations in pre-mRNA processing factors (PRPFs) cause autosomal-dominant retinitis pigmentosa (RP), but it is unclear why mutations in ubiquitously expressed genes cause non-syndromic retinal disease. Here, we generate transcriptome profiles from RP11 (PRPF31-mutated) patient-derived retinal organoids and retinal pigment epithelium (RPE), as well as Prpf31+/- mouse tissues, which revealed that disrupted alternative splicing occurred for specific splicing programmes. Mis-splicing of genes encoding pre-mRNA splicing proteins was limited to patient-specific retinal cells and Prpf31+/- mouse retinae and RPE. Mis-splicing of genes implicated in ciliogenesis and cellular adhesion was associated with severe RPE defects that include disrupted apical - basal polarity, reduced trans-epithelial resistance and phagocytic capacity, and decreased cilia length and incidence. Disrupted cilia morphology also occurred in patient-derived photoreceptors, associated with progressive degeneration and cellular stress. In situ gene editing of a pathogenic mutation rescued protein expression and key cellular phenotypes in RPE and photoreceptors, providing proof of concept for future therapeutic strategies.