Transcriptome analysis by strand-specific sequencing of complementary DNA

High-throughput complementary DNA sequencing (RNA-Seq) is a powerful tool for whole-transcriptome analysis, supplying information about a transcript's expression level and structure. However, it is difficult to determine the polarity of transcripts, and therefore identify which strand is transcribed. Here, we present a simple cDNA sequencing protocol that preserves information about a transcript's direction. Using Saccharomyces cerevisiae and mouse brain transcriptomes as models, we demonstrate that knowing the transcript's orientation allows more accurate determination of the structure and expression of genes. It also helps to identify new genes and enables studying promoter-associated and antisense transcription. The transcriptional landscapes we obtained are available online

FOX-2 Dependent Splicing of Ataxin-2 Transcript Is Affected by Ataxin-1 Overexpression

Alternative splicing is a fundamental posttranscriptional mechanism for controlling gene expression, and splicing defects have been linked to various human disorders. The splicing factor FOX-2 is part of a main protein interaction hub in a network related to human inherited ataxias, however, its impact remains to be elucidated. Here, we focused on the reported interaction between FOX-2 and ataxin-1, the disease-causing protein in spinocerebellar ataxia type 1. In this line, we further evaluated this interaction by yeast-2-hybrid analyses and co-immunoprecipitation experiments in mammalian cells. Interestingly, we discovered that FOX-2 localization and splicing activity is affected in the presence of nuclear ataxin-1 inclusions. Moreover, we observed that FOX-2 directly interacts with ataxin-2, a protein modulating spinocerebellar ataxia type 1 pathogenesis. Finally, we provide evidence that splicing of pre-mRNA of ataxin-2 depends on FOX-2 activity, since reduction of FOX-2 levels led to increased skipping of exon 18 in ataxin-2 transcripts. Most striking, we observed that ataxin-1 overexpression has an effect on this splicing event as well. Thus, our results demonstrate that FOX-2 is involved in splicing of ataxin-2 transcripts and that this splicing event is altered by overexpression of ataxin-1

role of ataxin-2 in transcriptional regulation

Die Spinozerebelläre Ataxie Typ 2 ist eine neurodegenerative Erkrankung, welche autosomal dominant vererbt und durch eine expandierte CAG-Wiederholung im SCA2-Gen ausgelöst wird. Die genaue Funktion des SCA2-Genprodukts Ataxin-2 (ATXN2) ist noch nicht ganz verstanden. Aufgrund struktureller Eigenschaften sowie experimentellen Beobachtungen wird ATXN2 eine Rolle im RNA-Metabolismus, bei der Endozytose und bei der Organisation des Aktin-Zytoskeletts zugeschrieben. Erste Yeast-two-Hybrid-(Y2H)-Analysen mit ATXN2 resultierten in der Identifizierung des transkriptionellen Regulators ZBRK1 (BRCA1-interacting protein with a KRAB domain 1) als potentiellen Interaktionspartner von ATXN2, welcher an der Reprimierung von Genen beteiligt ist. Weitergehende Analysen konnten die Assozierung beider Proteine mittels Y2H- und Ko- Immunopräzipitations-Experimenten bestätigen. Mikroskopische Analysen zeigten eine Ko-Lokalisierung beider Proteine im Zellkern. Interessanterweise konnte eine Korrelation zwischen erhöhter sowie reduzierter ZBRK1- und ATXN2-Expression beobachtet werden, was die Bildung eines regulatorischen Komplex‘ bestehend aus ZBRK1 und ATXN2 vermuten ließ. Bioinformatische Analysen mit dem bekannten ZBRK1-Bindemotiv führten zur Identifizierung potentieller ZBRK1-Bindemotive im SCA2-Promotor, welche durch Chromatin- Immunopräzipitation(ChIP)-, Promotoranalysen sowie durch Generierung eines spezifischen Intrabodys mit ZBRK1/ATXN2-interferierenden Eigenschaften charakterisiert wurden. Bemerkenswerterweise konnte zum ersten Mal gezeigt werden, dass ATXN2 als Ko-Regulator von ZBRK1 an der Aktivierung der eigenen SCA2-Transkription beteiligt ist, und somit den ersten Ko-Aktivator von ZBRK1 darstellt. Durch kombinierte bioinformatische Analysen und ATXN2-Defizienz- Experimente, konnte ein neues Zielgen des Komplexes, die Serine/Threonine- Kinase ATM (Ataxia telangiectasia mutated), identifiziert- sowie eine Aktivierung der ATM-Transkription durch den ZBRK1/ATXN2-Komplex mittels ChIP- bzw. Promotoranalysen bestätigt werden. Erste Hinweise aus Y2H-Analysen ließen eine Abnahme der ZBRK1/ATXN2-Interaktion mit zunehmender Polyglutaminlänge im ATXN2-Protein (ATXN2Q79) vermuten, welche wiederum anhand einer geringeren SCA2- bzw. ATM-Promotoraktivität durch die Expression von ATXN2Q79 bestätigt werden konnte. Abschließend konnte in Zellen, welche ATXN2Q79 exprimierten und mit oxidativem Stress behandelt wurden, eine veränderte Phoshorylierung der ATM-Substrate beobachtet werden, welche eine mögliche Konsequenz der beeinträchtigten ZBRK1/ATXN2Q79-Aktivierung von ATM sein könnte. Zusammenfassend konnte zum ersten Mal eine Beteiligung von ATXN2 in der transkriptionellen Aktivierung von Genen gezeigt werden. Interessanterweise beeinträchtigt die pathogene Polyglutaminexpansion diese Aktivierung des ATM- Promotors Demzufolge könnte eine Kombination aus transkriptioneller Dysregulation und einer veränderten Phosphorylierung der ATM- Substrate einen potentiellen Mechanismus in der Pathogenese von SCA2 darstellen.Spinocerebellar ataxia type 2 is an autosomal dominant inherited neurodegenerative disease, which is caused by an expanded CAG-repeat in the SCA2 gene. The cellular function of the SCA2 gene product Ataxin-2 (ATXN2) is not yet entirely understood. Protein function prediction and experimental studies have involved ATXN2 in RNA metabolism, endocytosis and in the organisation of the actin-cytoskeleton. Yeast-to-hybrid (Y2H) and co- immunoprecipitation assays have shown that ATXN2 interacts with ZBRK1 (BRCA1-interacting protein with a KRAB domain 1) and, microscopic analyses have shown co-localisation of both proteins in the nucleus. Over-expression and Knock-down experiments exhibited a correlation between ZBRK1 and ATXN2 transcripts levels, suggesting that a complex comprising ZBRK1 and ataxin-2 might regulate SCA2 gene transcription. Furthermore, bioinformatic analysis using the known ZBRK1 consensus DNA-binding motif revealed ZBRK1 binding sites in the SCA2 promoter. These predicted sites were experimentally validated by chromatin-immunoprecipitation, promoter-reporter assays and, disruption of ZBRK1/ATXN2 interaction by intrabody over-expression. Our results confirm that ataxin-2 acts as a co-regulator of ZBRK1 activating its own transcription. This work shows evidence for the first ZBRK1 co-activator described in the literature. Thereafter a combination of bioinformatic analysis and perturbation experiments resulted in the identification of a new target: the serin/threonin kinase ATM (Ataxia telangiectasia mutated). Predicted ZBRK1-binding sites in the ATM promoter were confirmed by chromatin- immunoprecipitation experiments. Promoter-reporter analyses showed an elevated ATM-promoter-activity when ZBRK1 and ATXN2 were over-expressed, illustrating a transcriptional activation of ATM through the ZBRK1/ATXN2 complex. Interestingly, ATXN2Q79 over-expression reduced the levels of both SCA2 and ATM promoter activity and caused a modified phosphorylation pattern on ATM substrates under oxidative stress conditions. Consequently, transcriptional deregulation and impaired phosphorylation of ATM substrates might constitute a potential mechanism in the pathogenesis of SCA2

Schematic model of FOX-2 and ATXN1 effects on ATXN2 transcript.

<p>The <i>SCA2</i> gene bears two putative FOX-binding sites downstream of exon 18 in the ATXN2 transcript as illustrated. Under normal conditions, FOX-2 binding resulted in inclusion of exon 18, whereas depletion of FOX-2 or overexpression of ATXN1 resulted in increased levels of ATXN2 transcripts lacking exon 18.</p

Co-localization of FOX-2 with nuclear ATXN1 inclusions is independent of the polyglutamine region.

<p>Confocal microscopy of HeLa cells transfected with CFP-ATXN1-Q0, CFP-ATXN1-Q30 or CFP-ATXN1-Q82, respectively. Forty-eight hours post transfection cells were fixed and prepared for microscopic analyses. FOX-2 protein was visualized using a specific antibody (Abnova). CFP fluorescence was pseudo-coloured green. Nuclei were stained using Hoechst. Bars represent 20 µm.</p

ATXN1 interacts with FOX-2 splice variants.

<p>(<b>A</b>) Schematic view of ATXN1 and the regions used in the Y2H analyses (left) and FOX-2 variants (right). Prey plasmids pACT-ATXN1-NTQ30 or pACT-ATXN1-NTQ82 cover amino acids 1–576, pACT-ATXN1-AXH amino acids 559–701, and pACT-ATXN1-CT amino acids 530–816. Bait plasmid pBTM-FOX-2_V1 covers amino acids 1–380 of variant 1, including the putative NLS in the C-terminal region, and pBTM-FOX-2_cyt covers amino acids 1–391 of the cytoplasmic FOX-2 variant. Different C-terminal regions of both FOX-2 variants are highlighted in blue and green. (<b>B; C</b>) For directed Y2H analyses yeast strain L40ccua was co-transformed with the relevant bait and prey plasmids, and transformants were selected and spotted onto selective media or membrane to analyze activity of the reporter genes. (<b>D</b>) For Co-IP experiments, HEK293T cell lysates derived from cells overexpressing FLAG-ATXN1-Q30 (left panel) or HEK293T cell lysates (right panel) were incubated with an antibody directed against FOX-2 (Bethyl or Abnova, respectively). Cell lysates incubated without primary antibody served as controls. Then, membranes were treated with an antibody directed against the FLAG tag or ATXN1 to detect precipitated protein.</p

Parent-reported impact of the COVID-19 pandemic on children and adolescents with ASD

The COVID-19 pandemic and its corresponding measures to prevent the spread of the virus have impacted us all, possible in particular youth with a pre-existing diagnosis of autism spectrum disorder (ASD). This study aimed to investigate the impact of the COVID-19 pandemic in children with ASD. Pre- and during COVID-19 data were collected. Data on ASD characteristics (SRS-2), and emotional and behavioral data (BPM-P) were available of respectively 39 and 31 participants. Results showed no significant difference with regard to ASD characteristics over time. However, the COVID-19 pandemic resulted in an increase in emotional and behavioral problems. Parents reported an increase in internalizing, externalizing, and attention problems. Boys seem to be particularly vulnerable to develop more internalizing problems

The FOX-2 splice variants FOX-2_V1 and FOX-2_cyt co-localize with nuclear ATXN1 inclusions.

<p>Confocal microscopy of HeLa cells transfected with (<b>A</b>) pCMV-HA-FOX-2_V1 and pcDNA1-FLAG-SCA1-Q30 or pcDNA1-FLAG-SCA1-Q82, or (<b>B</b>) pCMV-MYC-FOX-2_cyt and pcDNA1-FLAG-SCA1-Q30 or pcDNA1-FLAG-SCA1-Q82, and (<b>C</b>) pCMV-MYC-TIAR and pcDNA1-FLAG-SCA1-Q30 or pcDNA1-FLAG-SCA1-Q82, respectively. Forty-eight hours post transfection cells were fixed and prepared for microscopic analyses. Proteins were visualized using the respective antibodies against the tag as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037985#s4" target="_blank">Material and Methods</a>. Nuclei were stained using Hoechst. Bars represent 20 µm.</p

FOX-2 co-localizes with nuclear ATXN1 inclusions.

<p>(<b>A</b>) Endogenous localization of FOX-2 in HeLa cells visualized with an antibody directed against FOX-2 (Bethyl). (<b>B; C</b>) HeLa cells expressing normal and mutant ATXN1 were fixed forty-eight hours post transfection. Endogenous proteins were visualized using respective antibodies against (<b>B</b>) FOX-2 (Bethyl) and FLAG or (<b>C</b>) TIAR and FLAG as described. Nuclei were stained using Hoechst. Bars represent 20 µm.</p

ATXN2 interacts with FOX-2 splice variants and co-localizes with nuclear ATXN1 inclusions upon overexpression.

<p>(<b>A</b>) Schematic illustration of ATXN1 interactions. Black lines represent interactions reported by Lim and co-workers <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037985#pone.0037985-Lim1" target="_blank">[16]</a>, the blue line represents the investigated interaction in this study. (<b>B</b>) Schematic view of ATXN2 regions used in Y2H studies as described earlier <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037985#pone.0037985-Ralser1" target="_blank">[44]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037985#pone.0037985-Nonhoff1" target="_blank">[51]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037985#pone.0037985-Ralser2" target="_blank">[66]</a>. (<b>C; D</b>) L40ccua yeast cells expressing the corresponding LexA-ATXN2 and AD-FOX-2 fusion proteins were spotted onto selective media or membrane as indicated and the activity of the reporter genes was monitored. (<b>E</b>) HeLa cell lysates were incubated with an antibody directed against FOX-2 (Bethyl) and membranes were treated with an anti-ATXN2 antibody (BD-Biosciences) to detect precipitated protein. (<b>F</b>) HeLa cells were transfected with pCMV-MYC-ATXN2-Q22 or co-transfected with pCMV-MYC-ATXN2-Q22 and pcDNA1-FLAG-SCA1-Q30 or pcDNA1-FLAG-SCA1-Q82, respectively. Forty-eight hours post transfection cells were fixed and prepared for microscopic analyses. Nuclei were stained using Hoechst. Bars represent 20 µm.</p