Arabidopsis thaliana XRN2 is required for primary cleavage in the pre-ribosomal RNA

Three Rat1/Xrn2 homologues exist in Arabidopsis thaliana: nuclear AtXRN2 and AtXRN3, and cytoplasmic AtXRN4. The latter has a role in degrading 3′ products of miRNA-mediated mRNA cleavage, whereas all three proteins act as endogenous post-transcriptional gene silencing suppressors. Here we show that, similar to yeast nuclear Rat1, AtXRN2 has a role in ribosomal RNA processing. The lack of AtXRN2, however, does not result in defective formation of rRNA 5′-ends but inhibits endonucleolytic cleavage at the primary site P in the pre-rRNA resulting in the accumulation of the 35S* precursor. This does not lead to a decrease in mature rRNAs, as additional cleavages occur downstream of site P. Supplementing a P-site cleavage-deficient xrn2 plant extract with the recombinant protein restores processing activity, indicating direct participation of AtXRN2 in this process. Our data suggest that the 5′ external transcribed spacer is shortened by AtXRN2 prior to cleavage at site P and that this initial exonucleolytic trimming is required to expose site P for subsequent endonucleolytic processing by the U3 snoRNP complex. We also show that some rRNA precursors and excised spacer fragments that accumulate in the absence of AtXRN2 and AtXRN3 are polyadenylated, indicating that these nucleases contribute to polyadenylation-dependent nuclear RNA surveillance

Heat Stress Enhances the Accumulation of Polyadenylated Mitochondrial Transcripts in Arabidopsis thaliana

Background: Polyadenylation of RNA has a decisive influence on RNA stability. Depending on the organisms or subcellular compartment, it either enhances transcript stability or targets RNAs for degradation. In plant mitochondria, polyadenylation promotes RNA degradation, and polyadenylated mitochondrial transcripts are therefore widely considered to be rare and unstable. We followed up a surprising observation that a large number of mitochondrial transcripts are detectable in microarray experiments that used poly(A)-specific RNA probes, and that these transcript levels are significantly enhanced after heat treatment. Methodology/Principal Findings: As the Columbia genome contains a complete set of mitochondrial genes, we had to identify polymorphisms to differentiate between nuclear and mitochondrial copies of a mitochondrial transcript. We found that the affected transcripts were uncapped transcripts of mitochondrial origin, which were polyadenylated at multiple sites within their 39region. Heat-induced enhancement of these transcripts was quickly restored during a short recovery period. Conclusions/Significance: Our results show that polyadenylated transcripts of mitochondrial origin are more stable than previously suggested, and that their steady-state levels can even be significantly enhanced under certain conditions. As many microarrays contain mitochondrial probes, due to the frequent transfer of mitochondrial genes into the genome

Expanding the MicroRNA Targeting Code: Functional Sites with Centered Pairing

Most metazoan microRNA (miRNA) target sites have perfect pairing to the seed region, located near the miRNA 5′ end. Although pairing to the 3′ region sometimes supplements seed matches or compensates for mismatches, pairing to the central region has been known to function only at rare sites that impart Argonaute-catalyzed mRNA cleavage. Here, we present “centered sites,” a class of miRNA target sites that lack both perfect seed pairing and 3′-compensatory pairing and instead have 11–12 contiguous Watson-Crick pairs to the center of the miRNA. Although centered sites can impart mRNA cleavage in vitro (in elevated Mg[superscript 2+]), in cells they repress protein output without consequential Argonaute-catalyzed cleavage. Our study also identified extensively paired sites that are cleavage substrates in cultured cells and human brain. This expanded repertoire of cleavage targets and the identification of the centered site type help explain why central regions of many miRNAs are evolutionarily conserved.National Institutes of Health (U.S.)Damon Runyon Cancer Research Foundation. Fellowship Awar

A comprehensive expression profile of microRNAs and other classes of non-coding small RNAs in barley under phosphorous-deficient and -sufficient conditions

Phosphorus (P) is essential for plant growth. MicroRNAs (miRNAs) play a key role in phosphate homeostasis. However, little is known about P effect on miRNA expression in barley (Hordeum vulgare L.). In this study, we used Illumina's next-generation sequencing technology to sequence small RNAs (sRNAs) in barley grown under P-deficient and P-sufficient conditions. We identified 221 conserved miRNAs and 12 novel miRNAs, of which 55 were only present in P-deficient treatment while 32 only existed in P-sufficient treatment. Total 47 miRNAs were significantly differentially expressed between the two P treatments (|log₂| > 1). We also identified many other classes of sRNAs, including sense and antisense sRNAs, repeat-associated sRNAs, transfer RNA (tRNA)-derived sRNAs and chloroplast-derived sRNAs, and some of which were also significantly differentially expressed between the two P treatments. Of all the sRNAs identified, antisense sRNAs were the most abundant sRNA class in both P treatments. Surprisingly, about one-fourth of sRNAs were derived from the chloroplast genome, and a chloroplast-encoded tRNA-derived sRNA was the most abundant sRNA of all the sRNAs sequenced. Our data provide valuable clues for understanding the properties of sRNAs and new insights into the potential roles of miRNAs and other classes of sRNAs in the control of phosphate homeostasis.Michael Hackenberg, Po-Jung Huang, Chun-Yuan Huang, Bu-Jun Shi, Perry Gustafson and Peter Langridg

Comparative genomics of small RNA regulatory pathway components in vector mosquitoes

<p>Abstract</p> <p>Background</p> <p>Small RNA regulatory pathways (SRRPs) control key aspects of development and anti-viral defense in metazoans. Members of the Argonaute family of catalytic enzymes degrade target RNAs in each of these pathways. SRRPs include the microRNA, small interfering RNA (siRNA) and PIWI-type gene silencing pathways. Mosquitoes generate viral siRNAs when infected with RNA arboviruses. However, in some mosquitoes, arboviruses survive antiviral RNA interference (RNAi) and are transmitted via mosquito bite to a subsequent host. Increased knowledge of these pathways and functional components should increase understanding of the limitations of anti-viral defense in vector mosquitoes. To do this, we compared the genomic structure of SRRP components across three mosquito species and three major small RNA pathways.</p> <p>Results</p> <p>The <it>Ae. aegypti, An. gambiae </it>and <it>Cx. pipiens </it>genomes encode putative orthologs for all major components of the miRNA, siRNA, and piRNA pathways. <it>Ae. aegypti </it>and <it>Cx. pipiens </it>have undergone expansion of Argonaute and PIWI subfamily genes. Phylogenetic analyses were performed for these protein families. In addition, sequence pattern recognition algorithms MEME, MDScan and Weeder were used to identify upstream regulatory motifs for all SRRP components. Statistical analyses confirmed enrichment of species-specific and pathway-specific cis-elements over the rest of the genome.</p> <p>Conclusion</p> <p>Analysis of Argonaute and PIWI subfamily genes suggests that the small regulatory RNA pathways of the major arbovirus vectors, <it>Ae. aegypti and Cx. pipiens</it>, are evolving faster than those of the malaria vector <it>An. gambiae </it>and <it>D. melanogaster</it>. Further, protein and genomic features suggest functional differences between subclasses of PIWI proteins and provide a basis for future analyses. Common UCR elements among SRRP components indicate that 1) key components from the miRNA, siRNA, and piRNA pathways contain NF-kappaB-related and Broad complex transcription factor binding sites, 2) purifying selection has occurred to maintain common pathway-specific elements across mosquito species and 3) species-specific differences in upstream elements suggest that there may be differences in regulatory control among mosquito species. Implications for arbovirus vector competence in mosquitoes are discussed.</p

Evidence for Antisense Transcription Associated with MicroRNA Target mRNAs in Arabidopsis

Antisense transcription is a pervasive phenomenon, but its source and functional significance is largely unknown. We took an expression-based approach to explore microRNA (miRNA)-related antisense transcription by computational analyses of published whole-genome tiling microarray transcriptome and deep sequencing small RNA (smRNA) data. Statistical support for greater abundance of antisense transcription signatures and smRNAs was observed for miRNA targets than for paralogous genes with no miRNA cleavage site. Antisense smRNAs were also found associated with MIRNA genes. This suggests that miRNA-associated “transitivity” (production of small interfering RNAs through antisense transcription) is more common than previously reported. High-resolution (3 nt) custom tiling microarray transcriptome analysis was performed with probes 400 bp 5′ upstream and 3′ downstream of the miRNA cleavage sites (direction relative to the mRNA) for 22 select miRNA target genes. We hybridized RNAs labeled from the smRNA pathway mutants, including hen1-1, dcl1-7, hyl1-2, rdr6-15, and sgs3-14. Results showed that antisense transcripts associated with miRNA targets were mainly elevated in hen1-1 and sgs3-14 to a lesser extent, and somewhat reduced in dcl11-7, hyl11-2, or rdr6-15 mutants. This was corroborated by semi-quantitative reverse transcription PCR; however, a direct correlation of antisense transcript abundance in MIR164 gene knockouts was not observed. Our overall analysis reveals a more widespread role for miRNA-associated transitivity with implications for functions of antisense transcription in gene regulation. HEN1 and SGS3 may be links for miRNA target entry into different RNA processing pathways

Hypericum sp.: essential oil composition and biological activities

Phytochemical composition of Hypericum genus has been investigated for many years. In the recent past, studies on the essential oils (EO) of this genus have been progressing and many of them have reported interesting biological activities. Variations in the EO composition of Hypericum species influenced by seasonal variation, geographic distribution, phenological cycle and type of the organ in which EO are produced and/or accumulated have also been reported. Although many reviews attributed to the characterization as well as biological activities of H. perforatum crude extracts have been published, no review has been published on the EO composition and biological activities of Hypericum species until recently (Crockett in Nat Prod Commun 5(9):1493–1506, 2010; Bertoli et al. in Global Sci Books 5:29–47, 2011). In this article, we summarize and update information regarding the composition and biological activities of Hypericum species EO. Based on experimental work carried out in our laboratory we also mention possible biotechnology approaches envisaging EO improvement of some species of the genus.Fundação para a Ciência e a Tecnologia (FCT) - project PTDC/AGR AAM/70418/2006, SFRH/BD/ 13283/2003

RISC-interacting clearing 3’- 5’ exoribonucleases (RICEs) degrade uridylated cleavage fragments to maintain functional RISC in Arabidopsis thaliana

RNA-induced silencing complex (RISC) is composed of miRNAs and AGO proteins. AGOs use miRNAs as guides to slice target mRNAs to produce truncated 5’ and 3’ RNA fragments. The 5’ cleaved RNA fragments are marked with uridylation for degradation. Here, we identified novel cofactors of Arabidopsis AGOs, named RICE1 and RICE2. RICE proteins specifically degraded single-strand (ss) RNAs in vitro; but neither miRNAs nor miRNA*s in vivo. RICE1 exhibited a DnaQ-like exonuclease fold and formed a homohexamer with the active sites located at the interfaces between RICE1 subunits. Notably, ectopic expression of catalytically-inactive RICE1 not only significantly reduced miRNA levels; but also increased 5’ cleavage RISC fragments with extended uridine tails. We conclude that RICEs act to degrade uridylated 5’ products of AGO cleavage to maintain functional RISC. Our study also suggests a possible link between decay of cleaved target mRNAs and miRNA stability in RISC

Uridylation and adenylation of RNAs

The posttranscriptional addition of nontemplated nucleotides to the 3′ ends of RNA molecules can have a significant impact on their stability and biological function. It has been recently discovered that nontemplated addition of uridine or adenosine to the 3′ ends of RNAs occurs in different organisms ranging from algae to humans, and on different kinds of RNAs, such as histone mRNAs, mRNA fragments, U6 snRNA, mature small RNAs and their precursors etc. These modifications may lead to different outcomes, such as increasing RNA decay, promoting or inhibiting RNA processing, or changing RNA activity. Growing pieces of evidence have revealed that such modifications can be RNA sequence-specific and subjected to temporal or spatial regulation in development. RNA tailing and its outcomes have been associated with human diseases such as cancer. Here, we review recent developments in RNA uridylation and adenylation and discuss the future prospects in this research area

Antiviral Silencing and Suppression of Gene Silencing in Plants

RNA silencing is an evolutionary conserved sequence-specific gene inactivation mechanism that contributes to the control of development, maintains heterochromatin, acts in stress responses, DNA repair and defends against invading nucleic acids like transposons and viruses. In plants RNA silencing functions as one of the main immune systems. RNA silencing process involves the small RNAs and trans factor components like Dicers, Argonautes and RNA-dependent RNA poly- merases. To deal with host antiviral silencing responses viruses evolved mecha- nisms to avoid or counteract this, most notably through expression of viral suppressors of RNA silencing. Due to the overlap between endogenous and antiviral silencing pathways while blocking antiviral pathways viruses also impact endogenous silencing processes. Here we provide an overview of antiviral silencing pathway, host factors implicated in it and the crosstalk between antiviral and endogenous branches of silencing. We summarize the current status of knowledge about the viral counter-defense strategies acting at various steps during virus infection in plants with the focus on representative, well studied silencing suppres- sor proteins. Finally we discuss future challenges of the antiviral silencing and counter-defense research field