Autistic traits in myotonic dystrophy type 1 due to MBNL inhibition and RNA mis-splicing.

Tandem repeat expansions are enriched in autism spectrum disorder, including CTG expansion in the DMPK gene that underlines myotonic muscular dystrophy type 1. Although the clinical connection of autism to myotonic dystrophy is corroborated, the molecular links remained unknown. Here, we show a mechanistic path of autism via repeat expansion in myotonic dystrophy. We found that inhibition of muscleblind-like (MBNL) splicing factors by expanded CUG RNAs alerts the splicing of autism-risk genes during brain development especially a class of autism-relevant microexons. To provide in vivo evidence that the CTG expansion and MBNL inhibition axis leads to the presentation of autistic traits, we demonstrate that CTG expansion and MBNL-null mouse models recapitulate autism-relevant mis-splicing profiles and demonstrate social deficits. Our findings indicate that DMPK CTG expansion-associated autism arises from developmental mis-splicing. Understanding this pathomechanistic connection provides an opportunity for greater in-depth investigations of mechanistic threads in autism.

Allele-specific quantitation of ATXN3 and HTT transcripts in polyQ disease models.

BACKGROUND: The majority of genes in the human genome is present in two copies but the expression levels of both alleles is not equal. Allelic imbalance is an aspect of gene expression relevant not only in the context of genetic variation, but also to understand the pathophysiology of genes implicated in genetic disorders, in particular, dominant genetic diseases where patients possess one normal and one mutant allele. Polyglutamine (polyQ) diseases are caused by the expansion of CAG trinucleotide tracts within specific genes. Spinocerebellar ataxia type 3 (SCA3) and Huntington's disease (HD) patients harbor one normal and one mutant allele that differ in the length of CAG tracts. However, assessing the expression level of individual alleles is challenging due to the presence of abundant CAG repeats in the human transcriptome, which make difficult the design of allele-specific methods, as well as of therapeutic strategies to selectively engage CAG sequences in mutant transcripts. RESULTS: To precisely quantify expression in an allele-specific manner, we used SNP variants that are linked to either normal or CAG expanded alleles of the ataxin-3 (ATXN3) and huntingtin (HTT) genes in selected patient-derived cell lines. We applied a SNP-based quantitative droplet digital PCR (ddPCR) protocol for precise determination of the levels of transcripts in cellular and mouse models. For HD, we showed that the process of cell differentiation can affect the ratio between endogenous alleles of HTT mRNA. Additionally, we reported changes in the absolute number of the ATXN3 and HTT transcripts per cell during neuronal differentiation. We also implemented our assay to reliably monitor, in an allele-specific manner, the silencing efficiency of mRNA-targeting therapeutic approaches for HD. Finally, using the humanized Hu128/21 HD mouse model, we showed that the ratio of normal and mutant HTT transgene expression in brain slightly changes with the age of mice. CONCLUSIONS: Using allele-specific ddPCR assays, we observed differences in allele expression levels in the context of SCA3 and HD. Our allele-selective approach is a reliable and quantitative method to analyze low abundant transcripts and is performed with high accuracy and reproducibility. Therefore, the use of this approach can significantly improve understanding of allele-related mechanisms, e.g., related with mRNA processing that may be affected in polyQ diseases.

Implications of Poly(A) Tail Processing in Repeat Expansion Diseases.

Repeat expansion diseases are a group of more than 40 disorders that affect mainly the nervous and/or muscular system and include myotonic dystrophies, Huntington's disease, and fragile X syndrome. The mutation-driven expanded repeat tract occurs in specific genes and is composed of tri- to dodeca-nucleotide-long units. Mutant mRNA is a pathogenic factor or important contributor to the disease and has great potential as a therapeutic target. Although repeat expansion diseases are quite well known, there are limited studies concerning polyadenylation events for implicated transcripts that could have profound effects on transcript stability, localization, and translation efficiency. In this review, we briefly present polyadenylation and alternative polyadenylation (APA) mechanisms and discuss their role in the pathogenesis of selected diseases. We also discuss several methods for poly(A) tail measurement (both transcript-specific and transcriptome-wide analyses) and APA site identification-the further development and use of which may contribute to a better understanding of the correlation between APA events and repeat expansion diseases. Finally, we point out some future perspectives on the research into repeat expansion diseases, as well as APA studies.

Genetic tests based on the RT-PCR reaction in the diagnostics of SARS-CoV-2 infection.

INTRODUCTION: Since the SARS-CoV-2 emergence in 2019/2020, at least 158 million infections with this pathogen have been recorded, of which 3.29 million infected people have died. Due to the non-specific symptoms of SARS-CoV-2 infection, laboratory tests based on RT-PCR (reverse transcription and polymerase chain reaction) are mainly used in the diagnosis of COVID-19 disease. AIM: The aim of this study is to compare the molecular tests available on the Polish market for the diagnosis of SARS-CoV2 infection. RESULTS: Based on the data provided by the manufacturers and the performed laboratory analyses, we have shown that the available diagnostic kits differ mainly in the sensitivity and duration of the reaction. CONCLUSION: Due to the ongoing COVID-19 pandemic, the indicated parameters are key to effective control of the spread of SARS-CoV2, and therefore should be mainly taken into account when choosing and purchasing by diagnostic centres.

Artificial miRNAs targeting CAG repeat expansion in ORFs cause rapid deadenylation and translation inhibition of mutant transcripts.

Polyglutamine (polyQ) diseases are incurable neurological disorders caused by CAG repeat expansion in the open reading frames (ORFs) of specific genes. This type of mutation in the HTT gene is responsible for Huntington's disease (HD). CAG repeat-targeting artificial miRNAs (art-miRNAs) were shown as attractive therapeutic approach for polyQ disorders as they caused allele-selective decrease in the level of mutant proteins. Here, using polyQ disease models, we aimed to demonstrate how miRNA-based gene expression regulation is dependent on target sequence features. We show that the silencing efficiency and selectivity of art-miRNAs is influenced by the localization of the CAG repeat tract within transcript and the specific sequence context. Furthermore, we aimed to reveal the events leading to downregulation of mutant polyQ proteins and found very rapid activation of translational repression and HTT transcript deadenylation. Slicer-activity of AGO2 was dispensable in this process, as determined in AGO2 knockout cells generated with CRISPR-Cas9 technology. We also showed highly allele-selective downregulation of huntingtin in human HD neural progenitors (NPs). Taken together, art-miRNA activity may serve as a model of the cooperative activity and targeting of ORF regions by endogenous miRNAs.

RAN Translation of the Expanded CAG Repeats in the SCA3 Disease Context.

Spinocerebellar ataxia type 3 (SCA3) is a progressive neurodegenerative disorder caused by a CAG repeat expansion in the ATXN3 gene encoding the ataxin-3 protein. Despite extensive research the exact pathogenic mechanisms of SCA3 are still not understood in depth. In the present study, to gain insight into the toxicity induced by the expanded CAG repeats in SCA3, we comprehensively investigated repeat-associated non-ATG (RAN) translation in various cellular models expressing translated or non-canonically translated ATXN3 sequences with an increasing number of CAG repeats. We demonstrate that two SCA3 RAN proteins, polyglutamine (polyQ) and polyalanine (polyA), are found only in the case of CAG repeats of pathogenic length. Despite having distinct cellular localization, RAN polyQ and RAN polyA proteins are very often coexpressed in the same cell, impairing nuclear integrity and inducing apoptosis. We provide for the first time mechanistic insights into SCA3 RAN translation indicating that ATXN3 sequences surrounding the repeat region have an impact on SCA3 RAN translation initiation and efficiency. We revealed that RAN translation of polyQ proteins starts at non-cognate codons upstream of the CAG repeats, whereas RAN polyA proteins are likely translated within repeats. Furthermore, integrated stress response activation enhances SCA3 RAN translation. Our findings suggest that the ATXN3 sequence context plays an important role in triggering SCA3 RAN translation and that SCA3 RAN proteins may cause cellular toxicity.

A Functional Link Between Bir1 and the Saccharomyces cerevisiae Ctf19 Kinetochore Complex Revealed Through Quantitative Fitness Analysis.

The chromosomal passenger complex (CPC) is a key regulator of eukaryotic cell division, consisting of the protein kinase Aurora B/Ipl1 in association with its activator (INCENP/Sli15) and two additional proteins (Survivin/Bir1 and Borealin/Nbl1). Here, we report a genome-wide genetic interaction screen in Saccharomyces cerevisiae using the bir1-17 mutant, identifying through quantitative fitness analysis deletion mutations that act as enhancers and suppressors. Gene knockouts affecting the Ctf19 kinetochore complex were identified as the strongest enhancers of bir1-17, while mutations affecting the large ribosomal subunit or the mRNA nonsense-mediated decay pathway caused strong phenotypic suppression. Thus, cells lacking a functional Ctf19 complex become highly dependent on Bir1 function and vice versa. The negative genetic interaction profiles of bir1-17 and the cohesin mutant mcd1-1 showed considerable overlap, underlining the strong functional connection between sister chromatid cohesion and chromosome biorientation. Loss of some Ctf19 components, such as Iml3 or Chl4, impacted differentially on bir1-17 compared with mutations affecting other CPC components: despite the synthetic lethality shown by either iml3∆ or chl4∆ in combination with bir1-17, neither gene knockout showed any genetic interaction with either ipl1-321 or sli15-3 Our data therefore imply a specific functional connection between the Ctf19 complex and Bir1 that is not shared with Ipl1.

Structural Characteristics of Simple RNA Repeats Associated with Disease and their Deleterious Protein Interactions.

Short Tandem Repeats (STRs) are frequent entities in many transcripts, however, in some cases, pathological events occur when a critical repeat length is reached. This phenomenon is observed in various neurological disorders, such as myotonic dystrophy type 1 (DM1), fragile X-associated tremor/ataxia syndrome, C9orf72-related amyotrophic lateral sclerosis and frontotemporal dementia (C9ALS/FTD), and polyglutamine diseases, such as Huntington's disease (HD) and spinocerebellar ataxias (SCA). The pathological effects of these repeats are triggered by mutant RNA transcripts and/or encoded mutant proteins, which depend on the localization of the expanded repeats in non-coding or coding regions. A growing body of recent evidence revealed that the RNA structures formed by these mutant RNA repeat tracts exhibit toxic effects on cells. Therefore, in this review article, we present existing knowledge on the structural aspects of different RNA repeat tracts as revealed mainly using well-established biochemical and biophysical methods. Furthermore, in several cases, it was shown that these expanded RNA structures are potent traps for a variety of RNA-binding proteins and that the sequestration of these proteins from their normal intracellular environment causes alternative splicing aberration, inhibition of nuclear transport and export, or alteration of a microRNA biogenesis pathway. Therefore, in this review article, we also present the most studied examples of abnormal interactions that occur between mutant RNAs and their associated proteins.

Identifying proteins that bind to specific RNAs - focus on simple repeat expansion diseases.

RNA-protein complexes play a central role in the regulation of fundamental cellular processes, such as mRNA splicing, localization, translation and degradation. The misregulation of these interactions can cause a variety of human diseases, including cancer and neurodegenerative disorders. Recently, many strategies have been developed to comprehensively analyze these complex and highly dynamic RNA-protein networks. Extensive efforts have been made to purify in vivo-assembled RNA-protein complexes. In this review, we focused on commonly used RNA-centric approaches that involve mass spectrometry, which are powerful tools for identifying proteins bound to a given RNA. We present various RNA capture strategies that primarily depend on whether the RNA of interest is modified. Moreover, we briefly discuss the advantages and limitations of in vitro and in vivo approaches. Furthermore, we describe recent advances in quantitative proteomics as well as the methods that are most commonly used to validate robust mass spectrometry data. Finally, we present approaches that have successfully identified expanded repeat-binding proteins, which present abnormal RNA-protein interactions that result in the development of many neurological diseases.

Quantitative fitness analysis shows that NMD proteins and many other protein complexes suppress or enhance distinct telomere cap defects.

To better understand telomere biology in budding yeast, we have performed systematic suppressor/enhancer analyses on yeast strains containing a point mutation in the essential telomere capping gene CDC13 (cdc13-1) or containing a null mutation in the DNA damage response and telomere capping gene YKU70 (yku70Δ). We performed Quantitative Fitness Analysis (QFA) on thousands of yeast strains containing mutations affecting telomere-capping proteins in combination with a library of systematic gene deletion mutations. To perform QFA, we typically inoculate 384 separate cultures onto solid agar plates and monitor growth of each culture by photography over time. The data are fitted to a logistic population growth model; and growth parameters, such as maximum growth rate and maximum doubling potential, are deduced. QFA reveals that as many as 5% of systematic gene deletions, affecting numerous functional classes, strongly interact with telomere capping defects. We show that, while Cdc13 and Yku70 perform complementary roles in telomere capping, their genetic interaction profiles differ significantly. At least 19 different classes of functionally or physically related proteins can be identified as interacting with cdc13-1, yku70Δ, or both. Each specific genetic interaction informs the roles of individual gene products in telomere biology. One striking example is with genes of the nonsense-mediated RNA decay (NMD) pathway which, when disabled, suppress the conditional cdc13-1 mutation but enhance the null yku70Δ mutation. We show that the suppressing/enhancing role of the NMD pathway at uncapped telomeres is mediated through the levels of Stn1, an essential telomere capping protein, which interacts with Cdc13 and recruitment of telomerase to telomeres. We show that increased Stn1 levels affect growth of cells with telomere capping defects due to cdc13-1 and yku70Δ. QFA is a sensitive, high-throughput method that will also be useful to understand other aspects of microbial cell biology.

Involvement of the nuclear cap-binding protein complex in alternative splicing in Arabidopsis thaliana.

The nuclear cap-binding protein complex (CBC) participates in 5' splice site selection of introns that are proximal to the mRNA cap. However, it is not known whether CBC has a role in alternative splicing. Using an RT-PCR alternative splicing panel, we analysed 435 alternative splicing events in Arabidopsis thaliana genes, encoding mainly transcription factors, splicing factors and stress-related proteins. Splicing profiles were determined in wild type plants, the cbp20 and cbp80(abh1) single mutants and the cbp20/80 double mutant. The alternative splicing events included alternative 5' and 3' splice site selection, exon skipping and intron retention. Significant changes in the ratios of alternative splicing isoforms were found in 101 genes. Of these, 41% were common to all three CBC mutants and 15% were observed only in the double mutant. The cbp80(abh1) and cbp20/80 mutants had many more changes in alternative splicing in common than did cbp20 and cbp20/80 suggesting that CBP80 plays a more significant role in alternative splicing than CBP20, probably being a platform for interactions with other splicing factors. Cap-binding proteins and the CBC are therefore directly involved in alternative splicing of some Arabidopsis genes and in most cases influenced alternative splicing of the first intron, particularly at the 5' splice site.