Cristiane P G Calixto

University of São Paulo | USP · Institute of Bioscience (IB) (São Paulo)

RNA-sequencing (RNA-seq) is currently the method of choice for analysis of differential gene expression. To fully exploit the wealth of data generated from genome-wide transcriptomic approaches, the initial design of the experiment is of paramount importance. Biological rhythms in nature are pervasive and are driven by endogenous gene networks coll...

Background Accurate and comprehensive annotation of transcript sequences is essential for transcript quantification and differential gene and transcript expression analysis. Single molecule long read sequencing technologies provide improved integrity of transcript structures including alternative splicing, and transcription start and polyadenylatio...

A formação de biólogos baseia-se fortemente em um currículo presencial, incluindo inúmeras atividades práticas de laboratório e campo. Essa estrutura foi abruptamente abalada pela pandemia em 2020, forçando-nos a uma mudança para o ensino remoto emergencial (ERE). Sobrevivemos e nos adaptamos. Agora é hora de refletirmos com calma sobre a nossa prá...

Rice (Oryza sativa L.) is a major food crop but heat stress affects its yield and grain quality. To identify mechanistic solutions to improve rice yield under rising temperatures, molecular responses of thermotolerance must be understood. Transcriptional and post-transcriptional controls are involved in a wide range of plant environmental responses...

RNA-sequencing (RNA-seq) analysis of gene expression and alternative splicing should be routine and robust but is often a bottleneck for biologists because of different and complex analysis programs and reliance on specialized bioinformatics skills. We have developed the ‘3D RNA-seq’ App, an R shiny App and web-based pipeline for the comprehensive...

Alternative Splicing (AS) is a mechanism that generates different mature transcripts from precursor mRNAs (pre-mRNAs) of the same gene. In plants, a wide range of physiological and metabolic events are related to AS, as well as fast responses to changes in temperature. AS is present in around 60% of intron-containing genes in Arabidopsis, 46% in ri...

Alternative Splicing (AS) is a mechanism that generates different mature transcripts from precursor mRNAs (pre-mRNAs) of the same gene. In plants, a wide range of physiological and metabolic events are related to AS, as well as fast responses to changes in temperature. AS is present in around 60% of intron-containing genes in Arabidopsis, 46% in ri...

RNA-sequencing (RNA-seq) analysis of gene expression and alternative splicing should be routine and robust but is often a bottleneck for biologists because of different and complex analysis programs and reliance on skilled bioinformaticians to perform the analysis. To overcome these issues, we have developed the "3D RNA-seq" App, an R shiny App whi...

Plants re-program their gene expression when responding to changing environmental conditions. Besides differential gene expression, extensive alternative splicing (AS) of pre-mRNAs and changes in expression of long non-coding RNAs (lncRNAs) are associated with stress responses. RNA-sequencing of a diel time-series of the initial response of Arabido...

Assembly of the barley genome and extensive use of RNA-seq has resulted in an abundance of gene expression data and the recognition of wide-scale production of alternatively spliced transcripts. Here, we describe in detail a high-resolution reverse transcription-PCR based panel (HR RT-PCR) that confirms the accuracy of alternatively spliced transcr...

Table S1. Primers used in this study. Note S1: In silico pY and SUA binding site analysis of the LHY 5′UTR. Note S2: Schematics of primers locations within gene models. Note S3: Genotyping of mutant lines. Note S4: Descriptive statistics for best line fitting. Figure S1. Splicing factor gene structures and RNA‐seq isoform profiles. Figure S2....

Plants have adapted to tolerate and survive constantly changing environmental conditions by re-programming gene expression. The dynamics of the contribution of alternative splicing (AS) to stress responses are unknown. RNA-sequencing of a time-series of Arabidopsis thaliana plants exposed to cold determines the timing of significant AS changes. Thi...

One of the ways in which plants can respond to temperature is via alternative splicing (AS). Previous work showed that temperature changes affected the splicing of several circadian clock gene transcripts. Here we investigated the role of RNA-binding splicing factors (SFs) in temperature-sensitive alternative splicing (AS) of the clock gene LATE EL...

An alternative splicing isoform switch is where a pair of transcript isoforms reverse their relative expression abundances in response to external or internal stimuli. Although computational methods are available to study differential alternative splicing, few tools for detection of isoform switches exist and these are based on pairwise comparisons...

Background Co-expression has been widely used to identify novel regulatory relationships using high throughput measurements, such as microarray and RNA-seq data. Evaluation studies on co-expression network analysis methods mostly focus on networks of small or medium size of up to a few hundred nodes. For large networks, simulated expression data us...

Alternative splicing generates multiple transcript and protein isoforms from the same gene and thus is important in gene expression regulation. To date, RNA-sequencing (RNA-seq) is the standard method for quantifying changes in alternative splicing on a genome-wide scale. Understanding the current limitations of RNA-seq is crucial for reliable anal...

Alternative splicing (AS) is a regulated mechanism that generates multiple transcripts from individual genes. It is widespread in eukaryotic genomes and provides an effective way to control gene expression. At low temperatures, AS regulates Arabidopsis clock genes through dynamic changes in the levels of productive mRNAs. We examined AS in barley c...

Confirmation of NMD-impairment in the cycloheximide (CHX) assay by HR RT-PCR of HvELF3 transcripts. CHX treatment performed at both 20 and 4°C conditions. The barley line homozygous for the PTC-containing allele Hvelf3 (blue) showed increased abundance on CHX treatment (* represents P < 0.001) suggesting NMD is impaired upon CHX treatment. WT and B...

Barley gene-specific primers and their sequences used for expression and AS analyses. (PDF)

AS events in barley clock and clock-related genes detected by HR RT-PCR experiments. (PDF)

Report on some of the low abundance unproductive AS events in barley clock genes. (DOCX)

Sampling and temperature regime used in analyses of AS in regulating mRNA expression of clock genes. Sampling occurred 2.5 h after dawn. Red arrows represent time points when sampling occurred. Black boxes are for dark, white boxes are for light. (PDF)

Nucleotide alignment of HvPPD-H1 and HvPP73 Exon 6 with homologous regions in PRR3, PRR7, PRR37 and PRR73 from eight other plant species. AS events were identified at the 5’ and 3’ ends of exon 6 in barley. a Alignments of the 5’ end of exon 6 with flanking intron sequences and b the 3’ end of exon 6 with flanking intron sequences are shown. Alignm...

Cold-dependent LHY AS E6a transcripts detected by RT-PCR using primers located in E6 (forward) and E6a (reverse). Samples were harvested in the morning (2.5 h after dawn) in six different time-points/temperatures: 20°C (Day 7), Day 1 at 4°C, Day 2 at 4°C, 4°C (Day 4), Day 1 at 20°C and 20°C (Day 2). Amplification of UBC (HvUBC21) and PP2A (HvPP2AA2...

HR RT-PCR analysis of AS in the MYB-coding domain region of HvLHY. Exons are numbered on the genomic structure; 5’ UTR is the dark box; coding sequences are open boxes. Diagonal lines represent splicing events. AS events are shown in red. Approximate positions of primers are shown by arrowheads. Representative electropherograms of spectral data col...

Genomic structure and conserved AS events of Arabidopsis and barley GI and TOC1, and HvCO2. In the Arabidopsis genes, only those AS events which show conservation to barley are shown—for other Arabidopsis clock genes AS events see James et al. (2012a). Other abundant AS events in barley are also shown. Exons are numbered; 5’ and 3’ UTRs are dark bo...

Expression changes of clock genes during transfer from 20°C to 4°C. Total transcript levels of a HvLHY, b HvPPD-H1 and c HvPRR73 in the morning (2.5 h after dawn) at six different time-points/temperatures: 20°C (Day 7), Day 1 at 4°C, Day 2 at 4°C, 4°C (Day 4), Day 1 at 20°C and 20°C (Day 2). Results obtained with different HR RT-PCR primer pairs we...

Barley clock gene-specific primers used in AS analyses and their sequences. (PDF)

I. II. III. IV. V. VI. References SUMMARY: Re-programming of the transcriptome involves both transcription and alternative splicing (AS). Some genes are regulated only at the AS level with no change in expression at the gene level. AS data must be incorporated as an essential aspect of the regulation of gene expression. RNA-sequencing (RNA-seq) can...

After domestication, during a process of widespread range extension, barley adapted to a broad spectrum of agricultural environments. To explore how the barley genome responded to the environmental challenges it encountered, we sequenced the exomes of a collection of 267 georeferenced landraces and wild accessions. A combination of genome-wide anal...

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 splic...

Posttranscriptional control makes an important contribution to circadian regulation of gene expression. In higher plants, alternative splicing is particularly prevalent upon abiotic and biotic stress and in the circadian system. Here we describe in detail a high-resolution reverse transcription-PCR based panel (HR RT-PCR) to monitor alternative spl...

Table S1 Transcript complexity of AtRTD: distribution of the number of transcripts per gene

RNA-sequencing (RNA-seq) allows global gene expression analysis at the individual transcript level. Accurate quantification of transcript variants generated by alternative splicing (AS) remains a challenge. We have developed a comprehensive, nonredundant Arabidopsis reference transcript dataset (AtRTD) containing over 74 000 transcripts for use wit...

The circadian clock regulates a multitude of plant developmental and metabolic processes. In crop species, it contributes significantly to plant performance and productivity and to the adaptation and geographical range over which crops can be grown. To understand the clock in barley and how it relates to the components in the Arabidopsis thaliana c...

Small nucleolar RNAs (snoRNAs) and small Cajal body-specific RNAs (scaRNAs) are non-coding RNAs whose main function in eukaryotes is to guide the modification of nucleotides in ribosomal and spliceosomal small nuclear RNAs, respectively. Full-length sequences of Arabidopsis snoRNAs and scaRNAs have been obtained from cDNA libraries of capped and un...

The pistil, the female reproductive organ of plants, is a key player in the success of sexual plant reproduction. Ultimately, the production of fruits and seeds depends on the proper pistil development and function. Therefore, the identification and characterization of pistil expressed genes is essential for a better understanding and manipulation...