Arabidopsis microRNA expression regulation was studied in a wide array of abiotic stresses such as drought, heat, salinity, copper excess/deficiency, cadmium excess, and sulfur deficiency. A home-built RT-qPCR mirEX platform for the amplification of 289 Arabidopsis microRNA transcripts was used to study their response to abiotic stresses. Small RNA sequencing, Northern hybridization, and TaqMan® microRNA assays were performed to study the abundance of mature microRNAs. A broad response on the level of primary miRNAs (pri-miRNAs) was observed. However, stress response at the level of mature microRNAs was rather confined. The data presented show that in most instances, the level of a particular mature miRNA could not be predicted based on the level of its pri-miRNA. This points to an essential role of posttranscriptional regulation of microRNA expression. New Arabidopsis microRNAs responsive to abiotic stresses were discovered. Four microRNAs: miR319a/b, miR319b.2, and miR400 have been found to be responsive to several abiotic stresses and thus can be regarded as general stress-responsive microRNA species.

Liverworts are the most basal group of extant land plants. Nonetheless, the molecular biology of liverworts is poorly understood. Gene expression has been studied in only one species, Marchantia polymorpha. In particular, no microRNA (miRNA) sequences from liverworts have been reported.Here, Illumina-based next-generation sequencing was employed to identify small RNAs, and analyze the transcriptome and the degradome of Pellia endiviifolia.Three hundred and eleven conserved miRNA plant families were identified, and 42 new liverwort-specific miRNAs were discovered. The RNA degradome analysis revealed that target mRNAs of only three miRNAs (miR160, miR166, and miR408) have been conserved between liverworts and other land plants. New targets were identified for the remaining conserved miRNAs. Moreover, the analysis of the degradome permitted the identification of targets for 13 novel liverwort-specific miRNAs. Interestingly, three of the liverwort microRNAs show high similarity to previously reported miRNAs from Chlamydomonas reinhardtii.This is the first observation of miRNAs that exist both in a representative alga and in the liverwort P. endiviifolia but are not present in land plants. The results of the analysis of the P. endivifolia microtranscriptome support the conclusions of previous studies that placed liverworts at the root of the land plant evolutionary tree of life.

BackgroundIn the past few decades, non-coding RNAs (ncRNAs) have emerged as important regulators of gene expression in eukaryotes. Most studies of ncRNAs in plants have focused on the identification of silencing microRNAs (miRNAs) and small interfering RNAs (siRNAs). Another important family of ncRNAs that has been well characterized in plants is the small nucleolar RNAs (snoRNAs) and the related small Cajal body-specific RNAs (scaRNAs). Both target chemical modifications of ribosomal RNAs (rRNAs) and small nuclear RNAs (snRNAs). In plants, the snoRNA genes are organized in clusters, transcribed by RNA Pol II from a common promoter and subsequently processed into mature molecules. The promoter regions of snoRNA polycistronic genes in plants are highly enriched in two conserved cis-regulatory elements (CREs), Telo-box and Site II, which coordinate the expression of snoRNAs and ribosomal protein coding genes throughout the cell cycle.ResultsIn order to identify novel ncRNA genes, we have used the snoRNA Telo-box/Site II motifs combination as a functional promoter indicator to screen the Arabidopsis genome. The predictions generated by this process were tested by detailed exploration of available RNA-Seq and expression data sets and experimental validation. As a result, we have identified several snoRNAs, scaRNAs and 'orphan' snoRNAs. We also show evidence for 16 novel ncRNAs that lack similarity to any reported RNA family. Finally, we have identified two dicistronic genes encoding precursors that are processed to mature snoRNA and miRNA molecules. We discuss the evolutionary consequences of this result in the context of a tight link between snoRNAs and miRNAs in eukaryotes.ConclusionsWe present an alternative computational approach for non-coding RNA detection. Instead of depending on sequence or structure similarity in the whole genome screenings, we have explored the properties of promoter regions of well-characterized ncRNAs. Interestingly, besides expected ncRNAs predictions we were also able to recover single precursor arrangement for snoRNA-miRNA. Accompanied by analyses performed on rice sequences, we conclude that such arrangement might have interesting functional and evolutionary consequences and discuss this result in the context of a tight link between snoRNAs and miRNAs in eukaryotes.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-2221-x) contains supplementary material, which is available to authorized users.

tRNA-derived fragments (tRFs) constitute a new class of short regulatory RNAs that are a product of nascent or mature tRNA processing. tRF sequences have been identified in all domains of life; however, most published research pertains to human, yeast and some bacterial organisms. Despite growing interest in plant tRFs and accumulating evidence of their function in plant development and stress responses, no public, web-based repository dedicated to these molecules is currently available. Here, we introduce tRex (http://combio.pl/trex)-the first comprehensive data-driven online resource specifically dedicated to tRFs in the model plant Arabidopsis thaliana. The portal is based on verified Arabidopsis tRNA annotation and includes in-house-generated and publicly available small RNA sequencing experiments from various tissues, ecotypes, genotypes and stress conditions. The provided web-based tools are designed in a user-friendly manner and allow for seamless exploration of the data that are presented in the form of dynamic tables and cumulative coverage profiles. The tRex database is connected to external genomic and citation resources, which makes it a one-stop solution for Arabidopsis tRF-related research.

The Arabidopsis GUT15 RNA belongs to a class of noncoding RNAs that are expressed from the intergenic regions of protein-coding genes. We show that the RNA polymerase II transcribed GUT15 transcript serves as a precursor for two stable RNA species, a tRNA-like molecule and GUT15-tRF-F5, which are both encoded by the final intron in the GUT15 gene. The GUT15-encoded tRNA-like molecule cannot be autonomously transcribed by RNA polymerase III. However, this molecule contains a CCA motif, suggesting that it may enter the tRNA maturation pathway. The GUT15-encoded tRNA-like sequence has an inhibiting effect on the splicing of its host intron. Moreover, we demonstrate that the canonical tRNA genes nested within introns do not affect the splicing patterns of their host protein-coding transcripts.

FUS is a multifunctional protein involved in many steps of RNA metabolism, including transcription, splicing, miRNA processing and replication-dependent histone gene expression. In this paper, we show for the first time that FUS binds and negatively regulates the levels of a subset of snoRNAs in cells. Scanning of available human small RNA databases revealed the existence of smaller RNA fragments that can be processed from FUS-dependent snoRNAs. Therefore, we suggest that FUS mediates the biogenesis of snoRNA-derived small RNAs, called sdRNAs. Further in silico approaches enabled us to predict putative targets of selected FUS-dependent sdRNAs. Our results indicate that sdRNAs may bind to different regions of target mRNAs as well as to noncoding transcripts and influence the posttranscriptional level or translation of these targets.SIGNIFICANCE STATEMENTRNA metabolism is orchestrated by a complex network of RNA-protein interactions and involves various classes of RNA molecules. Small nucleolar RNAs (snoRNAs) are commonly considered essential components of the ribosome biogenesis pathway. However, recent studies have revealed that snoRNAs can also be fragmented into small entities called snoRNA-derived RNAs (sdRNAs), which have been linked to multiple cancer types and thus may serve as next-generation prognostic or diagnostic biomarkers. In this paper, a multifunctional protein, FUS, was shown to be involved in the biogenesis of snoRNA-derived fragments. Furthermore, we combined bioinformatic analyses with complementary experimental approaches to elucidate the role of FUS-dependent sdRNAs in gene expression regulation. Our findings reveal the considerable regulatory potential of this new class of small noncoding RNAs.

FUS is a multifunctional protein involved in many aspects of RNA metabolism, including transcription, splicing, translation, miRNA processing, and replication-dependent histone gene expression. In this work, we show that FUS depletion results in the differential expression of numerous small nucleolar RNAs (snoRNAs) that guide 2’-O methylation (2’-O-Me) and pseudouridylation of specific positions in ribosomal RNAs (rRNAs) and small nuclear RNAs (snRNAs). Using RiboMeth-seq and HydraPsiSeq for the profiling of 2’-O-Me and pseudouridylation status of rRNA species, we demonstrated considerable hypermodification at several sites in HEK293T and SH-SY5Y cells with FUS knockout (FUS KO) compared to wild-type cells. We observed a similar direction of changes in rRNA modification in differentiated SH-SY5Y cells with the FUS mutation (R495X) related to the severe disease phenotype of amyotrophic lateral sclerosis (ALS). Furthermore, the pattern of modification of some rRNA positions was correlated with the abundance of corresponding guide snoRNAs in FUS KO and FUS R495X cells. Our findings reveal a new role for FUS in modulating the modification pattern of rRNA molecules, that in turn might generate ribosome heterogeneity and constitute a fine-tuning mechanism for translation efficiency/fidelity. Therefore, we suggest that increased levels of 2’-O-Me and pseudouridylation at particular positions in rRNAs from cells with the ALS-linked FUS mutation may represent a possible new translation-related mechanism that underlies disease development and progression.

Drought is one of the main climate threats limiting crop production. Potato is one of the four most important food crop species worldwide and is sensitive to water shortage. The CBP80 gene was shown to affect Arabidopsis and potato responses to drought by regulating the level of microRNA159 and, consequently, the levels of the MYB33 and MYB101 transcription factors (TFs). Here, we show that three MYB TFs, MYB33, MYB65, and MYB101, are involved in plant responses to water shortage.Their downregulation in Arabidopsis causes stomatal hyposensitivity to abscisic acid (ABA), leading to reduced tolerance to drought. Transgenic Arabidopsis and potato plants overexpressing these genes, with a mutated recognition site in miR159, show hypersensitivity to ABA and relatively high tolerance to drought conditions. Thus, the MYB33, MYB65, and MYB101 genes may be potential targets for innovative breeding to obtain crops with relatively high tolerance to drought.