Impact of URT1-mediated uridylation on small RNA tailing in Arabidopsis
ABSTRACT: To analyse the impact of URT1-mediated uridylation on miRNA and siRNA tailing, we deep-sequenced small RNA libraries for WT and urt1 duplicate samples at the same developmental stage that was analyzed by TAIL-seq, i.e., two-week-old seedlings. Examination of miRNA and siRNA tailing in WT and urt1 samples.
Project description:To obtain a global view of mRNA uridylation in Arabidopsis, we generated TAIL-seq libraries from WT plants, urt1 and xrn4 single mutants, and urt1 xrn4 double mutant. The TAIL-seq protocol was recently developed to deep-sequence the 3' ends of RNAs (Chang et al., 2014). We generated TAIL-seq libraries from WT plants, urt1 and xrn4 single mutants, and urt1 xrn4 double mutant.
Project description:Small RNAs play essential regulatory roles in genome stability, development and stress responses in most eukaryotes. Plants encode DICER-LIKE (DCL) RNaseIII enzymes, including DCL1, which produces miRNAs, and DCL2, DCL3 and DCL4, which produce diverse size classes of siRNA. Plants also encode RNASE THREE-LIKE (RTL) enzymes that lack DCL-specific domains and whose function is largely unknown. Small RNA sequencing in plants over-expressing RTL1 or RTL2 or lacking RTL2 revealed that RTL1 over-expression inhibits the accumulation of all types of small RNAs produced by DCL2, DCL3 and DCL4, indicating that RTL1 is a general suppressor of plant siRNA pathways. By contrast, RTL2 plays minor, if any, role in the small RNA repertoire. In vivo and in vitro assays revealed that RTL1 prevents siRNA production by degrading dsRNA before they are processed by DCL2, DCL3 and DCL4. The substrate of RTL1 cleavage is likely long perfect (or near-perfect) dsRNA, consistent with the RTL1-insensitivity of miRNAs, which derive from short imperfect dsRNA. RTL1 is naturally expressed only weakly in roots, but virus infection strongly induces its expression in leaves, suggesting that RTL1 induction is a general strategy used by viruses to counteract the siRNA-based plant antiviral defense. Accordingly, transgenic plants over-expressing RTL1 are more sensitive to TYMV infection than wild-type plants, likely because RTL1 prevents the production of antiviral siRNAs. However, TCV, TVCV and CMV, which encode stronger suppressors of RNA silencing (VSR) than TYMV, are insensitive to RTL1 over-expression. Indeed, TCV VSR inhibits RTL1 activity, suggesting that inducing RTL1 expression and dampening RTL1 activity is a dual strategy used by viruses to establish a successful infection. These results reveal another level of complexity in the evolutionary battle between viruses and plant defenses. Flower sRNA profiles in diverse conditions involving RTL1 and RTL2
Project description:miRNAs are small non-coding RNAs that inhibit translation and promote mRNA decay. The levels of mature miRNAs are the result of different rates of transcription, processing, and turnover. The non-canonical polymerase Gld2 has been implicated in the stabilization of miR-122 possibly by catalyzing 3’ monoadenylation, however, there is little evidence that this relationship is one of cause and effect. Here, we biochemically characterize Gld2 involvement in miRNA monoadenylation and its effect on miRNA stability. We find that Gld2 directly monoadenylates and stabilizes specific miRNA populations in human fibroblasts and that sensitivity to monoadenylation-induced stability depends on nucleotides in the miRNA 3‘ end. These results establish a novel mechanism of miRNA stability and resulting post-transcriptional gene regulation. Sequencing of miRNAs to assess amount and 3' end monoadenylation state upon Gld2 knock-down.
Project description:In angiosperms, endosperm plays a crucial role in coordinating seed development through genetic balance and molecular interaction, and is the primary tissue where genomic imprinting occurs. To identify small interfering RNA (siRNA) “imprintome” and its paternal transcriptome activation in early developing maize endosperms, we performed high-throughput small RNA sequencing of whole kernels at 0, 3 and 5 days after pollination (DAP) and endosperms at 7, 10 and 15 DAP, using B73 by Mo17 reciprocal crosses. We observed gradually increased expression of paternal siRNAs in 3- and 5-DAP kernels and balanced contribution of parental siRNA transcriptome in 7-DAP endosperm, followed by identification 460 imprinted siRNA loci with majority (456/460, 99.1%) being maternally expressed that occurred at 10 DAP. Genome-wide scanning found 13 imprinted genes harboring imprinted siRNA loci within their 2-Kb flanking regions, which was significantly different from random probability based on simulation analysis. Finally, gene ontology categories of “response to auxin stimulus”, “response to brassinosteroid stimulus” and “regulation of gene expression” for genes harboring 10-DAP specific siRNAs and “nutrient reservoir activity”, “protein localization to vacuole” and “secondary metabolite biosynthetic process” for genes harboring 15-DAP specific siRNAs indicated that siRNAs could be involved in influencing specific cellular or biochemical processes that are essential for endosperm development, e.g. nutrient uptake and allocation. Although the mechanism of how these siRNAs regulating endosperm key events remains unclear, this study provided us an alternative perspective of siRNA function in plant. The unpollinated kernels (0 DAP), the kernels of 3, 5 DAP and endosperms of 7 10, 15 DAP from the B73 and Mo17 reciprocal crosses were used to perform high-throughput sequencing using the Illumina HiSeq2000 platform
Project description:During axon pathfinding, growth cones commonly exhibit changes in sensitivity to guidance cues that follow a strict timetable, even in the absence of pathway feedback, implicating cell-intrinsic regulation. Cellular timer mechanisms, however, are poorly understood. Here we have investigated microRNAs in the timing control of Sema3A sensitivity in retinal ganglion cell (RGC) growth cones. A developmental profiling screen identified miR-124 as a candidate timer. Loss of miR-124 delayed the onset of Sema3A sensitivity and concomitant Neuropilin-1 (NRP-1) receptor expression, and caused cell autonomous pathfinding errors. CoREST, a cofactor of a NRP1 repressor, was identified as a novel target and mediator of miR-124 for this highly specific temporal aspect of RGC growth cone responsiveness. Our findings indicate that miR-124 plays an important role in regulating the intrinsic temporal changes in RGC growth cone sensitivity and suggest that microRNAs may play a broad role as linear timers in vertebrate neuronal development. Two independent experiments were performed. One with a single sample for each of 3 stages, and the second with 2 biological replicates of each stage.
Project description:Most angiosperm nuclear DNA is repetitive and derived from silenced transposable elements (TEs). TE silencing requires substantial resources from the plant host, including the production of small interfering RNAs (siRNAs). Thus, the interaction between TEs and siRNAs is a critical aspect of both the function and the evolution of plant genomes. Yet the co-evolutionary dynamics between these two entities remains poorly characterized. Here we studied the organization of TEs within the maize (Zea mays ssp mays) genome, documenting that TEs fall within three groups based on the class and copy numbers. These groups included DNA elements, low copy RNA elements and higher copy RNA elements. The three groups varied statistically in characteristics that included length, location, age, siRNA expression and 24:22 nucleotide (nt) siRNA targeting ratios. In addition, the low copy retroelements encompassed a set of TEs that had previously been shown to decrease expression within a 24 nt siRNA biogenesis mutant (mop1). To investigate the evolutionary dynamics of the three groups, we estimated their abundance in two landraces, one with a genome similar in size to that of the maize reference and the other with a 30% larger genome. For all three accessions, we assessed TE abundance as well as 22 nt and 24 nt siRNA content within leaves. The high copy number retroelements are under targeted similarly by siRNAs among accessions, appear to be born of a rapid bust of activity, and may be currently transpositionally dead or limited. In contrast, the lower copy number group of retrolements are targeted more dynamically and have had a long and ongoing history of transposition in the maize genome We compared TE abundances and siRNA profiles among B73 (maize inbred line) and two additional maize landraces, Palomero Toluqueño (PT) and Olote Colorado (OAXA) using HTS
Project description:Animal germ cells employ small RNA-based mechanisms to recognize and silence DNA that invades their genome. One of these pathways is named the Piwi:piRNA pathway. Biogenesis of piRNAs is poorly understood. In C. elegans, the piRNA (21U-RNA)-binding Argonaute protein PRG-1 is the only known player acting downstream of pre-cursor transcription. From a screen aimed at the isolation of ‘piRNA-induced silencing defective’ mutations we identified, amongst known Piwi-pathway components like MUT-7, RDE-3 and HRDE-1, PID-1 as a novel player. PID-1 is essential for 21U RNA biogenesis and affects an early step in the processing or transport of 21U precursor transcripts. 12 small RNA samples were analyzed as singletons.
Project description:Alternative polyadenylation is an important cellular mechanism that enables generation of mRNA isoforms that differ in their 3' untranslated regions (3' UTRs) and consequently in their susceptibility to miRNA and RNA binding protein mediated regulation. A dramatic change in polyadenylation site usage, leading to the systematic expression of short 3’ UTR isoforms is known to occur upon induction of proliferation in resting cells. To understand the functional consequences of short 3’ UTR isoform expression we used 3' end sequencing and quantitative mass spectroscopy to determine polyadenylation site use, mRNA and protein levels in murine and human naive and activated T cells. We found that while the process and its impact on the susceptibility to miRNA and RNA binding protein mediated regulation are evolutionarily conserved, the conservation is poor at the level of individual orthologous genes. Contrary to the common belief, we did not find that transcriptome-wide 3' UTR shortening leads to a matched increase in mRNA and protein levels of genes with tandem polyadenylation sites. 3' ends of transcripts were profiled by high-throughput sequencing in murine and human naive and activated T cells.
Project description:MicroRNAs (miRNAs) are processed from longer precursors with fold-back structures. While animal MIRNA precursors have homogenous structures, plant precursors comprise a collection of fold-backs with variable size and shape. Here, we design an approach (SPARE) to systematically analyze miRNA processing intermediates and characterize the biogenesis of most of the evolutionary conserved miRNAs present in Arabidopsis thaliana. We found that plant MIRNAs are processed by four mechanisms, depending on the sequential direction of the processing machinery and the number of cuts required to release the miRNA. Classification of the precursors according to their processing mechanism revealed specific structural determinants for each group. We found that the complexity of the miRNA processing pathways occurs in both ancient and evolutionary young sequences, and that members of the same family can be processed in different ways. We observed that different structural determinants compete for the processing machinery and that alternative miRNAs can be generated from a single precursor. The results provide a mechanistic explanation for the structural diversity of MIRNA precursors in plants and new insights towards the understanding of the biogenesis of small RNAs. Approach to systematically analyze miRNA processing intermediates and characterize the biogenesis of conserved and young miRNAs present in Arabidopsis thaliana. MiRNA processing intermediates profiles of Wild type and Fiery mutants Arabidopsis plants were analyzed, using Illumina GAIIx.
Project description:Recent advances in genome-wide techniques allowed the identification of thousands of non-coding RNAs with various sizes in eukaryotes, some of which have further been shown to serve important functions in many biological processes. However, in model plant Arabidopsis, novel intermediate-sized ncRNAs (im-ncRNAs) (50~300nt) have very limited information. By using a modified isolation strategy combined with deep-sequencing technology, we identified 838 im-ncRNAs in Arabidopsis globally. More than half (58%) are new ncRNA species, mostly evolutionary divergent. Interestingly, annotated protein-coding genes with 5’-UTR derived novel im-ncRNAs tend to be highly expressed. For intergenic im-ncRNAs, their average abundances were comparable to mRNAs in seedlings, but subsets exhibited significantly lower expression in senescing leaves. Further, intergenic im-ncRNAs were regulated by similar genetic and epigenetic mechanisms as those of protein-coding genes, and some showed developmentally-regulated expression patterns. Large-scale reverse genetic screening showed that the down-regulation of a number of im-ncRNAs resulted in either obvious molecular changes or abnormal developmental phenotypes in vivo, indicating the functional importance of im-ncRNAs in plant growth and development. Together, our results demonstrate that novel Arabidopsis im-ncRNAs are developmentally-regulated and functional components discovered in the transcriptome. Genome-wide maps of Intermediate-size Non-coding RNAs in Arabidopsis