Project description:Expression profiles of MicroRNA and SiRNA of Arabidopsis thaliana Col-0 and transgenic plants with constitutive expression of the chimeric receptors NRG1 grown at different temperature To reveal the underlying molecular mechanism of de-cosuppression with memory by high temperature in Arabidopsis, we performed the expression profiles of microRNA and SiRNA in transgenic plants with constitutive expression of the chimeric receptors NRG1 and wide type Col-0 grown at different temperature using the Custom LC Sciences Arabidopsis microRNA and SiRNA array. Keywords: high temperature, de-cosuppression, MicroRNA, SiRNA
Project description:The secondary structure of an RNA molecule plays an integral role in its maturation, regulation, processing, and functionality. However, the global influence of this feature on plant gene expression is still for the most part unclear. Here, we use a high-throughput, sequencing-based, structure-mapping approach in conjunction with transcriptome-wide sequencing of polyA+-selected (RNA-seq), small (smRNA-seq), and ribosome-bound (ribo-seq) RNA populations to investigate the impact of RNA secondary structure on gene expression regulation in Arabidopsis. From this analysis, we find that highly unpaired and paired RNAs are strongly correlated with euchromatic and heterochromatic epigenetic histone modifications, respectively, providing further evidence that secondary structure is necessary for RNA-mediated posttranscriptional regulatory pathways. Additionally, we uncover key structural patterns across protein-coding transcripts that indicate RNA folding demarcates regions of protein translation and likely affects microRNA-mediated regulation of mRNAs in this model plant. We also reveal that RNA folding is significantly anti-correlated with overall transcript abundance, which is likely due to the increased propensity of highly structured mRNAs to be degraded and/or processed into smRNAs. Finally, we find that secondary structure affects mRNA translation, suggesting that this feature regulates plant gene expression at multiple levels. Overall, our findings provide the first global assessment of RNA folding and its significant regulatory effects in a plant transcriptome. Single-stranded RNA sequencing (ssRNA-seq) and ribosome-bound RNA sequencing (ribo-seq) in immature buds. A single replicate of each library.
Project description:Ribosomes translocate one codon at a time in mRNA during protein synthesis, but the regulators of ribosome translocation and their impact on neurodegenerative disease remain poorly understood. Here, we show that huntingtin (Htt) and its poly-Q expanded form, mHtt, a cause of Huntington disease (HD), promotes ribosome stalling and suppresses protein synthesis in vivo and in vitro. We found that fragile mental retardation protein (FMRP), a known promoter of ribosome stalling, is upregulated in HD cells and patients’ tissue. Unexpectedly, FMRP depletion fails to reverse mHtt-mediated ribosome stalling in HD cells. mHtt binds directly to ribosomes in a RNase-sensitive manner and interacts with ribosomal proteins. Combining high-resolution ribosome footprint profiling (Ribo-Seq) and RNA-Seq, we provide a global snapshot of stalling on mRNA transcripts, with a shift in ribosome occupancy toward the 5’ and 3’ end and single-codon unique pauses in HD cells. We also found ribosomes that appear to have stalled in mHtt mRNA before CAG repeat expansion. Thus, Htt regulates protein synthesis via ribosome stalling mechanisms and in turn may affect mRNA elongation, which may be exploited for HD therapeutics.
Project description:In plants, tasiRNAs form a class of endogenous secondary siRNAs produced through the action of RNA-DEPENDENT-RNA-POLYMERASE-6 (RDR6) upon microRNA-mediated cleavage of non-coding TAS RNAs. In Arabidopsis thaliana, TAS1, TAS2 and TAS4 tasiRNA production proceeds via a single cleavage event mediated by 22nt-long or/and asymmetric miRNAs in an ARGONAUTE-1 (AGO1)-dependent manner. By contrast, tasiRNA production from TAS3 seems to follow the so-called ‘two-hit’ process, where dual targeting of TAS3, specifically mediated by the 21nt-long, symmetric miR390, initiates AGO7-dependent tasiRNA production. Interestingly, features for TAS3 tasiRNA production differ in other plant species and we show here that such features also enable TAS3 tasiRNA biogenesis in Arabidopsis, and that a single miR390 targeting event is, in fact, sufficient for this process, suggesting that the ‘one-hit’ model underpins all the necessary rudiments of secondary siRNA biogenesis from plant TAS transcripts. Further results suggest that the two-hit configuration likely enhances the fidelity of tasiRNA production and, hence, the accuracy of downstream gene regulation. Finally, we show that a ‘non-cleavable one-hit’ process allows tasiRNA production from both TAS1 and TAS3 transcripts, indicating that RDR6 recruitment does not require miRNA cleavage, nor does the recruitment, as we further show, of SUPRRESSOR-OF-GENE-SILENCING-3, indispensable for tasiRNA generation.
Project description:Environmental stress is detrimental to plants viability and requires an adequate reprogramming of cellular activities to maximize plant survival. We present a global analysis of the adaptive stress response of Arabidopsis thaliana to prolonged heat stress. We combine deep sequencing of RNA and ribosome protected fragments to provide genome wide map of adaptation to heat stress on at transcriptional and translational level. Our analysis shows that the genes with the highest upregulation upon heat stress are known heat-responsive gene, chaperons and other genes involved in protein folding control. Majority of these genes exhibits increase on both transcriptional and translational level. No translational inhibition or ribosome stalling was observed, which can be observed in the early thermal stress response, indicating that plants alter their cellular composition in order to adapt to the prolonged exposure to increased temperatures.
Project description:We previously reported that ribosome stalling at AUG-stop sequences in the 5'-UTR plays a critical role in regulating the expression of Arabidopsis thaliana NIP5;1, which encodes a boron uptake transporter, in response to boron conditions in media. Here, we conducted ribosome profiling analysis to reveal the genome-wide regulation of translation in response to boron conditions in A. thaliana. We identified 460 translationally regulated genes. Transcripts with reduced translation efficiency were rich in upstream open reading frames (uORFs), highlighting the importance of uORF-mediated translational regulation. We found that 148 uORF instances had greater ribosome density under high boron conditions. Moreover, translationally downregulated transcripts were rich in minimum uORFs (AUG-stops), suggesting that AUG-stops play a global role in the boron response. Boron increased the ribosome occupancy of stop codons, indicating that this element is involved in global translational termination processes.
Project description:Dodders (Cuscuta spp.) are obligate parasitic plants that obtain water and nutrients from the stems of host plants via specialized feeding structures called haustoria. Dodder haustoria facilitate bi-directional movement of viruses, proteins, and mRNAs between host and parasite, but the functional effects of these movements are not clear. Here we show that C. campestris haustoria accumulate high levels of many novel microRNAs (miRNAs) while parasitizing Arabidopsis thaliana hosts. Many of these miRNAs are 22 nts long, a usually rare size of plant miRNA associated with amplification of target silencing through secondary small interfering RNA (siRNA) production. Several A. thaliana mRNAs are targeted by C. campestris 22 nt miRNAs during parasitism, resulting in high levels of secondary siRNA production. The targeted mRNAs function in hormone perception, pathogen-defense signaling, phloem function, and stem-cell identity. Homologs of these target mRNAs from diverse plants also have high-confidence complementary sites to C. campestris miRNAs, suggesting that homologous mRNAs are targeted by C. campestris across its very broad host range. These data show that C. campestris miRNAs act as trans-species regulators of host gene expression, and suggest that they may act as virulence factors during parasitism.