Andrea Barta | Medical University of Vienna and University of Vienna (original) (raw)

Papers by Andrea Barta

Proceedings in Life Sciences, 1984

The past years have seen tremendous progress in our knowledge of the structures of ribosomal prot... more The past years have seen tremendous progress in our knowledge of the structures of ribosomal proteins and RNA (for review articles see Wittmann 1983; Noller 1984, Wollenzien et al. 1984). Yet our understanding of the ribosomal mechanisms has remained rather fragmentary. The extremely complex interplay of the various factors involved in initiation, elongation, and termination, the interactions of mRNA and tRNA with the various ribosomal components, and finally the molecular processes involved in transpeptidation and translocation still represent a “terra incognita” in our concept of protein biosynthesis. However, techniques have been developed in the last few years which today allow us to focus on at least some of the reactions taking place on an actively translating ribosome (Spirin and Serdyuk, 1984). Chemical methods such as affinity and photoaffinity labelling techniques and cross-linking with bifunctional reagents or UV light have been employed to detect functional domains on the ribosome. In particular affinity and photoaffinity reactions have been used successfully to study the ribosomal binding sites for tRNA, mRNA, and antibiotics. The literature on this subject is too extensive to be discussed here in any detail. For summaries on the work performed the reader is referred to several review articles (Pellegrini and Cantor 1977; Kuchler 1978; Cantor 1979; Kuchler and Ofengand 1979; Johnson 1979; Ofengand 1980).

Molecular Biology Reports, 1987

To study the influence of the ubiquitous cap structure of nuclear pre-mRNAs on the assembly of a ... more To study the influence of the ubiquitous cap structure of nuclear pre-mRNAs on the assembly of a functional splicing complex, the in vitro splicing of a truncated human metallothionein pre-mRNA was examined in the presence of the cap analogue m7GTP. Significant inhibition of splicing was observed at a concentration as low as 5 pM m7GTP. Analysis of the splicing reaction on glycerol density gradients showed two complexes sedimenting at 45S apd 22S. When the reaction was carried out in presence of m'GTP a marked decrease of the material sedimenting at 45S, representing the active splicing complex, was observed. When capped pre-mRNA was replaced by uncapped pre-mRNA, complex formation was significantly reduced. These data indicate that the cap structure plays an important yet unknown role in the assembly of spliceosomes.

DNA, 1984

Expression of the rat growth hormone (rGH) gene in the pituitary and in cultured pituitary tumor ... more Expression of the rat growth hormone (rGH) gene in the pituitary and in cultured pituitary tumor cells is regulated by glucocorticoid hormones. After co-transfer of cloned DNA containing the rGH gene with the herpes simplex virus (HSV) thymidine kinase (tk) gene into mouse Ltk- cells, rGH gene transcripts were detected in eight of fifteen tk+ cell lines. However, in all eight clones, the predominant rGH gene transcript was only about 0.75 kb, 0.3 kb shorter than pituitary rGH mRNA. The 0.75-kb transcripts, examined from one clone, L-rGH-4, lacked sequences derived from exons 1 and 2 of the rGH gene. Although transcripts larger than 0.75 kb were detected, the normal 2.2-kb rGH gene primary transcript was present only at very low levels. Nuclease mapping studies also failed to reveal transcripts initiated at the normal rGH gene promoter, but instead revealed transcripts with 5&amp;amp;amp;#39; termini arising within intron B of the gene. These data suggest either that transcripts arise from internal promoters within the rGH gene or that a transcript initiated upstream from the normal promoter was processed abnormally. Dexamethasone increased the levels of the 0.75-kb rGH gene transcripts about fourfold in all eight clones expressing rGH mRNA. These data suggest that structural elements important for glucocorticoid-mediated influences on regulation of GH gene expression are contained within the transferred rGH gene fragment and can function even when the normal rGH gene promoter is not used and the pattern of expression is grossly abnormal.

Nucleic Acids Research, Dec 15, 2012

AtCyp59 is a multidomain cyclophilin containing a peptidyl-prolyl cis/trans isomerase (PPIase) do... more AtCyp59 is a multidomain cyclophilin containing a peptidyl-prolyl cis/trans isomerase (PPIase) domain and an evolutionarily highly conserved RRM domain. Deregulation of this class of cyclophilins has been shown to affect transcription and to influence phosphorylation of the C-terminal repeat domain of the largest subunit of the RNA polymerase II. We used a genomic SELEX method for identifying RNA targets of AtCyp59. Analysis of the selected RNAs revealed an RNA-binding motif (G[U/C]N[G/A]CC[A/G]) and we show that it is evolutionarily conserved. Binding to this motif was verified by gel shift assays in vitro and by RNA immunopreciptation assays of AtCyp59 in vivo. Most importantly, we show that binding also occurs on unprocessed transcripts in vivo and that binding of specific RNAs inhibits the PPIase activity of AtCyp59 in vitro. Surprisingly, genome-wide analysis showed that the RNA motif is present in about 70% of the annotated transcripts preferentially in exons. Taken together, the available data suggest that these cyclophilins might have an important function in transcription regulation.

Genome Research, May 1, 2015

Service Email Alerting click here. top right corner of the article or Receive free email alerts w... more Service Email Alerting click here. top right corner of the article or Receive free email alerts when new articles cite this article-sign up in the box at the http://genome.cshlp.org/subscriptions

Nucleic Acids Research, Nov 29, 2011

Alternative splicing (AS) coupled to nonsensemediated decay (NMD) is a post-transcriptional mecha... more Alternative splicing (AS) coupled to nonsensemediated decay (NMD) is a post-transcriptional mechanism for regulating gene expression. We have used a high-resolution AS RT-PCR panel to identify endogenous AS isoforms which increase in abundance when NMD is impaired in the Arabidopsis NMD factor mutants, upf1-5 and upf3-1. Of 270 AS genes (950 transcripts) on the panel, 102 transcripts from 97 genes (32%) were identified as NMD targets. Extrapolating from these data around 13% of intron-containing genes in the Arabidopsis genome are potentially regulated by AS/NMD. This cohort of naturally occurring NMD-sensitive AS transcripts also allowed the analysis of the signals for NMD in plants. We show the importance of AS in introns in 5 0 or 3 0 UTRs in modulating NMD-sensitivity of mRNA transcripts. In particular, we identified upstream open reading frames overlapping the main start codon as a new trigger for NMD in plants and determined that NMD is induced if 3 0-UTRs were >350 nt. Unexpectedly, although many intron retention transcripts possess NMD features, they are not sensitive to NMD. Finally, we have shown that AS/NMD regulates the abundance of transcripts of many genes important for plant development and adaptation including transcription factors, RNA processing factors and stress response genes.

Background Alternative splicing is the major post-transcriptional mechanism by which gene express... more Background Alternative splicing is the major post-transcriptional mechanism by which gene expression is regulated and affects a wide range of processes and responses in most eukaryotic organisms. RNA-sequencing (RNA-seq) can generate genome-wide quantification of individual transcript isoforms to identify changes in expression and alternative splicing. RNAseq is an essential modern tool but its ability to accurately quantify transcript isoforms depends on the diversity, completeness and quality of the transcript information. Results We have developed a new Reference Transcript Dataset for Arabidopsis (AtRTD2) for RNAseq analysis containing over 82k non-redundant transcripts, whereby 74,194 transcripts originate from 27,667 protein-coding genes. A total of 13,524 protein-coding genes have at least one alternatively spliced transcript in AtRTD2 such that about 60% of the 22,453 protein-coding, intron-containing genes in Arabidopsis undergo alternative splicing. More than 600 putative U12 introns were identified in more than 2,000 transc ripts. AtRTD2 was generated from transcript assemblies of ca. 8.5 billion pairs of reads from 285 RNA-seq data sets obtained from 129 RNA-seq libraries and merged along with the previous version, AtRTD, and Araport11 transcript assemblies. AtRTD2 increases the diversity of transcripts and through application of stringent filters represents the most extensive and accurate transcript collection for Arabidopsis to date. We have demonstrated a generally good correlation of alternative splicing ratios from RNA-seq data analysed by Salmon and experimental data from high resolution RT-PCR. However, we have observed inaccurate quantification of transcript isoforms for genes with multiple transcripts which have variation in the lengths of their UTRs. This variation is not effectively corrected in RNA-seq analysis programmes and will therefore impact RNA-seq analyses generally. To address this, we have tested different genome-wide modifications of AtRTD2 to improve transcript quantification and alternative splicing analysis. As a result, we release AtRTD2-QUASI specifically for use in Quantification of Alternatively Spliced Isoforms and demonstrate that it out-performs other available transcriptomes for RNA-seq analysis. Conclusions We have generated a new transcriptome resource for RNA-seq analyses in Arabidopsis (AtRTD2) designed to address quantification of different isoforms and alternative splicing in .

Journal of Biological Chemistry, Oct 1, 2002

Arg/Ser-rich (RS) proteins play a crucial role in splicing and are implicated in splice site sele... more Arg/Ser-rich (RS) proteins play a crucial role in splicing and are implicated in splice site selection in metazoa. In plants, intron recognition seems to differ from the one in animals due to specific factor requirements. Here we describe a new plant-specific RS-rich protein, atRSZ33, with a unique domain structure consisting of an RNA recognition motif (RRM), two zinc knuckles embedded in a basic RS region, and an acidic C-terminal domain. atRSZ33 was found to be a phosphoprotein that concentrates in nuclear speckles and is predominantly present in roots and flowers. In a yeast two-hybrid screen, atRSZ33 interacted with splicing factors atSRp34/SR1, an Arabidopsis ortholog of human SF2/ ASF; atRSZp21 and atRSZp22, which are similar to the human 9G8; and three novel SC35-like splicing factors termed atSCL28, atSCL30, and atSCL33/SR33. Two further members of the SCL family, namely SCL30a and the ortholog of mammalian SC35, atSC35, were also found to interact with atRSZ33. These interactions were verified by in vitro binding assays; furthermore, the transcriptional activity of atRSZ33 was found to overlap with the ones of its interacting partners. These specific interactions coupled with the many similarities of atRSZ33 to SR proteins suggest that its main activity is in spliceosome assembly. Mapping of regions necessary for protein-protein interaction between atRSZ33 and atSCL33/ SR33 revealed that both zinc knuckles together with a small part of the RS and the RRM domain are required for efficient binding. However, the interacting domain is relatively small, allowing binding of additional proteins, a feature that is consistent with the proposed role of atRSZ33 in spliceosome assembly.

DNA, Feb 1, 1987

Genomic clones containing the closely related genes for human growth hormone (hGH) and chorionic ... more Genomic clones containing the closely related genes for human growth hormone (hGH) and chorionic somatomammotropin (hCS) were obtained from genomic bacteriophage X and cosmid libraries. The entire GH/CS chromosomal locus was reconstructed utilizing overlapping restriction fragments characterized from the isolated clones. The hGH/hCS locus contains two GH genes and three CS genes spanning 48 kb of DNA in the order: 5'-(hGH-l/hCS-5/hCS-l/hGH-2/hCS-2)-3', confirming analysis of cosmid clones obtained from a different human library (Barsh et al., 1983). To complete the characterization of the hCS genes, the nucleotide sequence of the hCS-5 gene was determined. Sequence analysis revealed a mutation of the 5' splice site at the exon II-intron B boundary, suggesting that the hCS-5 gene is a pseudogene. The nucleotide sequence of an allelic variant of the hCS-2 gene was determined and found to contain a single amino acid substitution and the deletion of a single codon. The hGH/hCS gene locus was further characterized by the localization of at least 27 Alu-\\ pe repetitive sequences and identification of three unique sequences in the vicinity of several hGH and hCS genes which define the probable breakpoints of the evolutionary duplication units. These data, combined with the nucleotide sequences of all five GH and CS genes, indicate that the hGH/hCS gene locus has evolved by duplication mechanisms. Evidence for the occurrence of at least one gene conversion event involving the hCS-1 gene precursor and the hCS-2 gene was found, indicating that the hGH/hCS gene locus has evolved by concerted mechanisms. The structure of the hCS genes is discussed in light of recent studies of CS genes from other mammalian species.

Trends in Plant Science, Oct 1, 2012

Biochemical Society Transactions, May 21, 2008

The impact of AS (alternative splicing) is well-recognized in animal systems as a key regulator o... more The impact of AS (alternative splicing) is well-recognized in animal systems as a key regulator of gene expression and proteome complexity. In plants, AS is of growing importance as more genes are found to undergo AS, but relatively little is known about the factors regulating AS or the consequences of AS on mRNA levels and protein function. We have established an accurate and reproducible RT (reverse transcription)-PCR system to analyse AS in multiple genes. Initial studies have identified new AS events confirming that current values for the frequency of AS in plants are likely to be underestimates.

Current Topics in Microbiology and Immunology, 2008

SR proteins are a family of splicing factors important for splice site recognition and spliceosom... more SR proteins are a family of splicing factors important for splice site recognition and spliceosome assembly. Their ability to bind to RNA and to interact with proteins as well identifies them as important players in splice site choice and alternative splicing. Plants possess twice as many SR proteins as animals, and some of the subfamilies are plant specific. Arabidopsis SR proteins are involved in different aspects of plant growth and development as well as in responses to environmental cues. The plant-specific subfamilies have been shown to be regulated by alternative splicing events, which are highly conserved in evolution. The tight regulation of splicing factors by alternative splicing might allow coordinated responses of their target genes.

Journal of Biological Chemistry, Nov 1, 1984

The sequence of one of the two major expressed human chorionic somatomammotropin genes (hCS-1) wa... more The sequence of one of the two major expressed human chorionic somatomammotropin genes (hCS-1) was determined. The hCS-1 gene and the human growth hormone gene (hGH-1) share 92% nucleotide sequence homology in their 5'- and 3'-flanking regions, introns and exons. This finding, in addition to the existence of multiple closely linked hGH and hCS genes, suggests that these genes are evolving by concerted mechanisms. S1 nuclease, hybridization, and primer extension analysis of placental poly(A+) RNA demonstrated the presence of two functional initiation sites within the hCS-1 and/or the hCS-2 gene(s). The majority (about 95%) of the transcripts initiate 30 nucleotides downstream from the TATAAA sequence, and about 5% of the transcripts initiate 30 nucleotides downstream from a CATAAA sequence located 55 nucleotides 5' to the TATAAA sequence. The presence of two functional promoter elements as well as direct repeated sequences flanking the TATAAA sequence and exon I of the hCS genes are consistent with the hypothesis (Cooke, N. E., and Baxter, J. D. (1982) Nature (Lond.) 297, 603-606) that an important regulatory element may have been inserted into the gene in a separate evolutionary event. An analysis of other direct repeats, internal homology, and homology between other growth hormone and chorionic somatomammotropin genes offers a more extensive conceptualization of how this gene family evolved.

The Plant Cell, Oct 1, 2013

Alternative splicing (AS) of precursor mRNAs (pre-mRNAs) from multiexon genes allows organisms to... more Alternative splicing (AS) of precursor mRNAs (pre-mRNAs) from multiexon genes allows organisms to increase their coding potential and regulate gene expression through multiple mechanisms. Recent transcriptome-wide analysis of AS using RNA sequencing has revealed that AS is highly pervasive in plants. Pre-mRNAs from over 60% of intron-containing genes undergo AS to produce a vast repertoire of mRNA isoforms. The functions of most splice variants are unknown. However, emerging evidence indicates that splice variants increase the functional diversity of proteins. Furthermore, AS is coupled to transcript stability and translation through nonsense-mediated decay and microRNA-mediated gene regulation. Widespread changes in AS in response to developmental cues and stresses suggest a role for regulated splicing in plant development and stress responses. Here, we review recent progress in uncovering the extent and complexity of the AS landscape in plants, its regulation, and the roles of AS in gene regulation. The prevalence of AS in plants has raised many new questions that require additional studies. New tools based on recent technological advances are allowing genome-wide analysis of RNA elements in transcripts and of chromatin modifications that regulate AS. Application of these tools in plants will provide significant new insights into AS regulation and crosstalk between AS and other layers of gene regulation.

Plant Journal, Dec 15, 2007

Alternative splicing (AS) increases the proteomic and functional capacity of genomes through the ... more Alternative splicing (AS) increases the proteomic and functional capacity of genomes through the generation of alternative mRNA transcripts from the same gene. AS is now estimated to occur in a third of Arabidopsis and rice genes, and includes genes involved in the control of growth and development, responses to stress and signalling. Regulation of AS reflects the interactions between positive and negative cis sequences in the precursor messenger RNA and a range of trans-acting factors. The levels and activities of these factors differ in different cells and growth conditions. To identify changes in AS in multiple genes simultaneously, we have established a reproducible RT-PCR panel that can analyse 96 alternative splicing events and accurately measure the ratio of alternatively spliced products. This procedure detected statistically significant changes in AS in different plant organs, in plants grown under different light and day-length conditions, and in plants overexpressing splicing factors. The system provides a convenient, medium-throughput means of monitoring changes in AS in multiple genes. It can readily be applied to much larger or targeted sets of gene transcripts to generate information on the significance and regulation of AS in plant growth and development, specific processes and responses to external stimuli.

Planta, Oct 1, 1989

The effects of various concentrations of cadmium on Nicotiana tabacum L. cv. Xanthi suspension ce... more The effects of various concentrations of cadmium on Nicotiana tabacum L. cv. Xanthi suspension cells were examined. Surprisingly, certain concentrations of Cd (100 150gM) stimulated growth of cell cultures considerably, whereas all other concentrations were inhibitory. Synthesis of DNA was severly affected in a dose-dependent manner by Cd concentrations of 250 gM and higher. In contrast, RNA and protein synthesis were similarly stimulated by 100 gM Cd, thus indicating that enhancement of RNA synthesis was the primary cause for the observed stimulation of cell culture growth. The transient expression of a chimeric chloramphenicol-acetyltransferase gene was similarly affected by Cd. When the effects of other heavy metals (Cu, Zn, Pb, Co, Mn, A1) on these cellular processes were investigated, only Zn showed a comparable stimulation of RNA and protein synthesis, although a tenfold higher concentration of Zn compared with Cd was required. As Zn and Cd are chemically very similar, these results are discussed in view of the well-known role of Zn in the regulation of transcription.

RNA, Oct 1, 1998

Ribosomes are multifunctional RNP complexes whose catalytic activities absolutely depend on dival... more Ribosomes are multifunctional RNP complexes whose catalytic activities absolutely depend on divalent metal ions. It is assumed that structurally and functionally important metal ions are coordinated to highly ordered RNA structures that form metal ion binding pockets. One potent tool to identify the structural surroundings of high-affinity metal ion binding pockets is metal ion-induced cleavage of RNA. Exposure of ribosomes to divalent metal ions, such as Pb 2+ , Mg 2+ , Mn 2+ , and Ca 2+ , resulted in site-specific cleavage of rRNAs. Sites of strand scission catalyzed by different cations accumulate at distinct positions, indicating the existence of general metal ion binding centers in the highly folded rRNAs in close proximity to the cleavage sites. Two of the most efficient cleavage sites are located in the 59 domain of both 23S and 16S rRNA, regions that are known to self-fold even in the absence of ribosomal proteins. Some of the efficient cleavage sites were mapped to the peptidyl transferase center located in the large ribosomal subunit. Furthermore, one of these cleavages was clearly diminished upon AcPhe-tRNA binding to the P site, but was not affected by uncharged tRNA. This provides evidence for a close physical proximity of a metal ion to the amino acid moiety of charged tRNAs. Interestingly, comparison of the metal ion cleavage pattern of eubacterial 70S with that of human 80S ribosomes showed that certain cleavage sites are evolutionarily highly conserved, thus demonstrating an identical location of a nearby metal ion. This suggests that cations, bound to evolutionarily constrained binding sites, are reasonable candidates for being of structural or functional importance.

Nucleic Acids Research, Aug 26, 2006

Alternative splicing is an important mechanism for fine tuning of gene expression at the posttran... more Alternative splicing is an important mechanism for fine tuning of gene expression at the posttranscriptional level. SR proteins govern splice site selection and spliceosome assembly. The Arabidopsis genome encodes 19 SR proteins, several of which have no orthologues in metazoan. Three of the plant specific subfamilies are characterized by the presence of a relatively long alternatively spliced intron located in their first RNA recognition motif, which potentially results in an extremely truncated protein. In atRSZ33, a member of the RS2Z subfamily, this alternative splicing event was shown to be autoregulated. Here we show that atRSp31, a member of the RS subfamily, does not autoregulate alternative splicing of its similarily positioned intron. Interestingly, this alternative splicing event is regulated by atRSZ33. We demonstrate that the positions of these long introns and their capability for alternative splicing are conserved from green algae to flowering plants. Moreover, in particular alternative splicing events the splicing signals are embedded into highly conserved sequences. In different taxa, these conserved sequences occur in at least one gene within a subfamily. The evolutionary preservation of alternative splice forms together with highly conserved intron features argues for additional functions hidden in the genes of these plant-specific SR proteins.

Nucleic Acids Research, Dec 17, 2015

The formation of RNA-DNA hybrids, referred to as R-loops, can promote genome instability and canc... more The formation of RNA-DNA hybrids, referred to as R-loops, can promote genome instability and cancer development. Yet the mechanisms by which Rloops compromise genome instability are poorly understood. Here, we establish roles for the evolutionarily conserved Nrl1 protein in pre-mRNA splicing regulation, R-loop suppression and in maintaining genome stability. nrl1 mutants exhibit endogenous DNA damage, are sensitive to exogenous DNA damage, and have defects in homologous recombination (HR) repair. Concomitantly, nrl1 cells display significant changes in gene expression, similar to those induced by DNA damage in wild-type cells. Further, we find that nrl1 cells accumulate high levels of R-loops, which co-localize with HR repair factors and require Rad51 and Rad52 for their formation. Together, our findings support a model in which R-loop accumulation and subsequent DNA damage sequesters HR factors, thereby compromising HR repair at endogenously or exogenously induced DNA damage sites, leading to genome instability.