The Arabidopsis U1 snRNP regulates mRNA 3'-end processing.

The removal of introns by the spliceosome is a key gene regulatory mechanism in eukaryotes, with the U1 snRNP subunit playing a crucial role in the early stages of splicing. Studies in metazoans show that the U1 snRNP also conducts splicing-independent functions, but the lack of genetic tools and knowledge about U1 snRNP-associated proteins have limited the study of such splicing-independent functions in plants. Here we describe an RNA-centric approach that identified more than 200 proteins associated with the Arabidopsis U1 snRNP and revealed a tight link to mRNA cleavage and polyadenylation factors.

A high-resolution single-molecule sequencing-based Arabidopsis transcriptome using novel methods of Iso-seq analysis.

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 polyadenylation sites. However, accuracy is significantly affected by sequencing errors, mRNA degradation, or incomplete cDNA synthesis.

Independent parental contributions initiate zygote polarization in Arabidopsis thaliana.

Embryogenesis of flowering plants is initiated by polarization of the zygote, a prerequisite for correct axis formation in the embryo. The daughter cells of the asymmetric zygote division form the pro-embryo and the mostly extra-embryonic suspensor. The suspensor plays a pivotal role in nutrient and hormone transport and rapid growth of the embryo.

Transcriptome Sequence Reveals Candidate Genes Involving in the Post-Harvest Hardening of Trifoliate Yam .

Storage ability of trifoliate yam () is restricted by a severe post-harvest hardening (PHH) phenomenon, which starts within the first 24 h after harvest and renders tubers inedible. Previous work has only focused on the biochemical changes affecting PHH in . To the best of our knowledge, the candidate genes responsible for the hardening of have not been identified.

A novel isoform of cryptochrome 4 (Cry4b) is expressed in the retina of a night-migratory songbird.

The primary sensory molecule underlying light-dependent magnetic compass orientation in migratory birds has still not been identified. The cryptochromes are the only known class of vertebrate proteins which could mediate this mechanism in the avian retina. Cryptochrome 4 of the night-migratory songbird the European robin (Erithacus rubecula; erCry4) has several of the properties needed to be the primary magnetoreceptor in the avian eye.

Marchantia TCP transcription factor activity correlates with three-dimensional chromatin structure.

Information in the genome is not only encoded within sequence or epigenetic modifications, but is also found in how it folds in three-dimensional space. The formation of self-interacting genomic regions, named topologically associated domains (TADs), is known as a key feature of genome organization beyond the nucleosomal level. However, our understanding of the formation and function of TADs in plants is extremely limited.

Metabolic Labeling of RNAs Uncovers Hidden Features and Dynamics of the Arabidopsis Transcriptome.

Transcriptome analysis by RNA sequencing (RNA-seq) has become an indispensable research tool in modern plant biology. Virtually all RNA-seq studies provide a snapshot of the steady state transcriptome, which contains valuable information about RNA populations at a given time but lacks information about the dynamics of RNA synthesis and degradation. Only a few specialized sequencing techniques, such as global run-on sequencing, have been used to provide information about RNA synthesis rates in plants.

The U1 snRNP Subunit LUC7 Modulates Plant Development and Stress Responses via Regulation of Alternative Splicing.

Introns are removed by the spliceosome, a large macromolecular complex composed of five ribonucleoprotein subcomplexes (U snRNPs). The U1 snRNP, which binds to 5' splice sites, plays an essential role in early steps of the splicing reaction. Here, we show that LETHAL UNLESS CBC7 (LUC7) proteins, which are encoded by a three-member gene family in Arabidopsis, are important for plant development and stress resistance.

Arabidopsis RNA processing factor SERRATE regulates the transcription of intronless genes.

Intron splicing increases proteome complexity, promotes RNA stability, and enhances transcription. However, introns and the concomitant need for splicing extend the time required for gene expression and can cause an undesirable delay in the activation of genes. Here, we show that the plant microRNA processing factor SERRATE (SE) plays an unexpected and pivotal role in the regulation of intronless genes.

PORCUPINE regulates development in response to temperature through alternative splicing.

Recent findings suggest that alternative splicing has a critical role in controlling the responses of plants to temperature variations. However, alternative splicing factors in plants are largely uncharacterized. Here we establish the putative splice regulator, PORCUPINE (PCP), as temperature-specific regulator of development in Arabidopsis thaliana.

Determining Nucleosome Position at Individual Loci After Biotic Stress Using MNase-qPCR.

Nucleosome occupancy in promoter and genic regions can severely influence the transcription levels. Few methods have been established to investigate the nucleosome occupancy along the DNA. In this chapter we describe a detailed protocol to analyze the nucleosome occupancy at a specific locus using MNase-pPCR.

Rapid Assessment of DNA Methylation Changes in Response to Salicylic Acid by Chop-qPCR.

Methylation of cytosines plays an important role in epigenetic regulation of gene expression. Several methods exist to determine the methylation status of DNA. Here, we describe a rapid and cost-effective method called Chop-qPCR to determine dynamic changes in the DNA methylation patterns, as they occur for instance in response to environmental stresses.

Immunoprecipitation-based analysis of protein-protein interactions.

Several techniques allow the detection of protein-protein interactions. In vivo co-immunoprecipitation (Co-IP) studies are an important complement to other commonly used techniques such as yeast two-hybrid or fluorescence complementation, as they reveal interactions between functional proteins at physiological relevant concentrations. Here, we describe an in vivo Co-IP approach using either GFP affinity matrix or specific antibodies to purify proteins of interests and their interacting partners.

Rapid and parallel quantification of small and large RNA species.

Quantitative real-time PCR (qRT-PCR) is a common technique for mRNA quantification. Several methods have been developed in the past few years in order to adapt qRT-PCR also for small non-coding RNAs (sRNA). We here provide a simple and sensitive protocol that allows quantification of mRNAs, selected sRNAs, and long non-coding RNAs (lncRNA) in one cDNA sample by qRT-PCR.

RACK1 and the microRNA pathway: is it déjà-vu all over again?

MicroRNAs (miRNAs) control many aspects of development and adaption in plants and in animals by post-transcriptional control of mRNA stability and translatability. Over the last years numerous proteins have been identified in the miRNA pathway. The versatile scaffold protein RACK1 has been associated with efficient miRNA production and function in plants and metazoans.

RACK1 scaffold proteins influence miRNA abundance in Arabidopsis.

MicroRNAs (miRNAs) regulate plant development by post-transcriptional regulation of target genes. In Arabidopsis thaliana, DCL1 processes precursors (pri-miRNAs) to miRNA duplexes, which associate with AGO1. Additional proteins act in concert with DCL1 (e.

Enhanced microRNA accumulation through stemloop-adjacent introns.

MicroRNAs (miRNAs) originate from stemloop-forming precursor RNAs found in longer primary transcripts that often contain introns. We show that in plants, those introns, when located 3' of the stemloop, can promote mature miRNA accumulation, through a mechanism that likely operates at the level of miRNA processing or stability. Reversely, when miRNA production is reduced such as in dicer-like 1 mutants, splicing of introns that promote miRNA processing is considerably increased, pointing to a tight physical and temporal coordination of intron splicing and miRNA processing in plants.

Fast-forward genetics identifies plant CPL phosphatases as regulators of miRNA processing factor HYL1.

MicroRNAs (miRNAs) are processed from primary transcripts that contain partially self-complementary foldbacks. As in animals, the core microprocessor in plants is a Dicer protein, DICER-LIKE1 (DCL1). Processing accuracy and strand selection is greatly enhanced through the RNA binding protein HYPONASTIC LEAVES 1 (HYL1) and the zinc finger protein SERRATE (SE).

RNA 3' processing functions of Arabidopsis FCA and FPA limit intergenic transcription.

The RNA-binding proteins FCA and FPA were identified based on their repression of the flowering time regulator FLC but have since been shown to have widespread roles in the Arabidopsis thaliana genome. Here, we use whole-genome tiling arrays to show that a wide spectrum of genes and transposable elements are misexpressed in the fca-9 fpa-7 (fcafpa) double mutant at two stages of seedling development. There was a significant bias for misregulated genomic segments mapping to the 3' region of genes.

Support vector machines-based identification of alternative splicing in Arabidopsis thaliana from whole-genome tiling arrays.

Background: Alternative splicing (AS) is a process which generates several distinct mRNA isoforms from the same gene by splicing different portions out of the precursor transcript. Due to the (patho-)physiological importance of AS, a complete inventory of AS is of great interest. While this is in reach for human and mammalian model organisms, our knowledge of AS in plants has remained more incomplete.

Global effects of the small RNA biogenesis machinery on the Arabidopsis thaliana transcriptome.

In Arabidopsis thaliana, four different dicer-like (DCL) proteins have distinct but partially overlapping functions in the biogenesis of microRNAs (miRNAs) and siRNAs from longer, noncoding precursor RNAs. To analyze the impact of different components of the small RNA biogenesis machinery on the transcriptome, we subjected dcl and other mutants impaired in small RNA biogenesis to whole-genome tiling array analysis. We compared both protein-coding genes and noncoding transcripts, including most pri-miRNAs, in two tissues and several stress conditions.

MicroRNA gene evolution in Arabidopsis lyrata and Arabidopsis thaliana.

MicroRNAs (miRNAs) are short regulatory RNAs processed from partially self-complementary foldbacks within longer MIRNA primary transcripts. Several MIRNA families are conserved deeply through land plants, but many are present only in closely related species or are species specific. The finding of numerous evolutionarily young MIRNA, many with low expression and few if any targets, supports a rapid birth-death model for MIRNA evolution.

AGRONOMICS1: a new resource for Arabidopsis transcriptome profiling.

Transcriptome profiling has become a routine tool in biology. For Arabidopsis (Arabidopsis thaliana), the Affymetrix ATH1 expression array is most commonly used, but it lacks about one-third of all annotated genes present in the reference strain. An alternative are tiling arrays, but previous designs have not allowed the simultaneous analysis of both strands on a single array.

Stress-induced changes in the Arabidopsis thaliana transcriptome analyzed using whole-genome tiling arrays.

The responses of plants to abiotic stresses are accompanied by massive changes in transcriptome composition. To provide a comprehensive view of stress-induced changes in the Arabidopsis thaliana transcriptome, we have used whole-genome tiling arrays to analyze the effects of salt, osmotic, cold and heat stress as well as application of the hormone abscisic acid (ABA), an important mediator of stress responses. Among annotated genes in the reference strain Columbia we have found many stress-responsive genes, including several transcription factor genes as well as pseudogenes and transposons that have been missed in previous analyses with standard expression arrays.