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.

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.

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.

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.

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.

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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.

ACKNOWLEDGEMENTSThis work is dedicated to the memory of Professor Wlodzimierz J. Krzyzosiak. We thank Professor Marta Olejniczak from Department of Genome Engineering for reviewing the manuscript and every day stimulating discussions. We thank Professor Jerzy Ciesiolka from Department of RNA Biochemistry for helpful suggestions. The confocal microscopy analyses were performed in the ABSTRACT 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 noncognate codons upstream of the CAG repeats, whereas RAN polyA proteins are likely translated within repeats. Furthermore, integrated stress response activation enhances SCA3 RAN translation. We suggest that RAN translation in SCA3 is a common event substantially contributing to SCA3 pathogenesis and that the ATXN3 sequence context plays an important role in triggering this unconventional translation. KEYWORDSSCA3, CAG repeat expansion, RAN translation, translation initiation, non-cognate initiation codon, integrated stress response ABBREVIATIONS ATXN3 -ataxin-3, c9ALS/FTD -c9orf72 amyotrophic lateral sclerosis/frontotemporal dementia, CHOP -C/EBP homologous protein, DM1 -myotonic dystrophy type 1 and type 2, ER -endoplasmic reticulum, FECD -Fuchs' endothelial corneal dystrophy, FXTAS -fragile X tremor ataxia syndrome, iMet-tRNAinitiator Met-tRNA, ISR -integrated stress response, MetAPs -methionine aminopeptidases, NAT -Nterminal acetyltransferase, NPC -nuclear pore complex, polyApolyalanine, polyQpolyglutamine, polySpolyserine, RAN translation -repeat associated non-ATG translation, SA -sodium arsenite, SCA3 -spinocerebellar ataxia type 3, TG -thapsigargin INTRODUCTIONSpinocerebellar ataxia type 3 (SCA3) is the most common form of dominantly inherited ataxia in the world and one of nine polyglutamine (polyQ) neurodegenerative diseases. This progressive and fatal disorder is primarily characterized by neuronal dysfunction and degeneration of the cerebellum and functionall...