Identification of SMG6 cleavage sites and a preferred RNA cleavage motif by global analysis of endogenous NMD targets in human cells.

In metazoans, cleavage by the endoribonuclease SMG6 is often the first degradative event in non-sense-mediated mRNA decay (NMD). However, the exact sites of SMG6 cleavage have yet to be determined for any endogenous targets, and most evidence as to the identity of SMG6 substrates is indirect. Here, we use Parallel Analysis of RNA Ends to specifically identify the 5' termini of decay intermediates whose production is dependent on SMG6 and the universal NMD factor UPF1. In this manner, the SMG6 cleavage sites in hundreds of endogenous NMD targets in human cells have been mapped at high resolution. In addition, a preferred sequence motif spanning most SMG6 cleavage sites has been discovered and validated by mutational analysis. For many SMG6 substrates, depletion of SMG6 resulted in the accumulation of decapped transcripts, an effect indicative of competition between SMG6-dependent and SMG6-independent NMD pathways. These findings provide key insights into the mechanisms by which mRNAs targeted by NMD are degraded.

Comprehensive investigation of microRNAs enhanced by analysis of sequence variants, expression patterns, ARGONAUTE loading, and target cleavage.

MicroRNAs (miRNAs) are a class of small RNAs that typically function by guiding the cleavage of target messenger RNAs. They have been shown to play major roles in a variety of plant processes, including development, and responses to pathogens and environmental stresses. To identify new miRNAs and regulation in Arabidopsis (Arabidopsis thaliana), 27 small RNA libraries were constructed and sequenced from various tissues, stresses, and small RNA biogenesis mutants, resulting in 95 million genome-matched sequences. The use of rdr2 to enrich the miRNA population greatly enhanced this analysis and led to the discovery of new miRNAs arising from both known and new precursors, increasing the total number of Arabidopsis miRNAs by about 10%. Parallel Analysis of RNA Ends data provide evidence that the majority guide target cleavage. Many libraries represented novel stress/tissue conditions, such as submergence-stressed flowers, which enabled the identification of new stress regulation of both miRNAs and their targets, all of which were validated in wild-type plants. By combining small RNA expression analysis with ARGONAUTE immunoprecipitation data and global target cleavage data from Parallel Analysis of RNA Ends, a much more complete picture of Arabidopsis miRNAs was obtained. In particular, the discovery of ARGONAUTE loading and target cleavage biases gave important insights into tissue-specific expression patterns, pathogen responses, and the role of sequence variation among closely related miRNA family members that would not be evident without this combinatorial approach.

Evidence that XRN4, an Arabidopsis homolog of exoribonuclease XRN1, preferentially impacts transcripts with certain sequences or in particular functional categories.

One of the major players controlling RNA decay is the cytoplasmic 5'-to-3' exoribonuclease, which is conserved among eukaryotic organisms. In Arabidopsis, the 5'-to-3' exoribonuclease XRN4 is involved in disease resistance, the response to ethylene, RNAi, and miRNA-mediated RNA decay. Curiously, XRN4 appears to display selectivity among its substrates because certain 3' cleavage products formed by miRNA-mediated decay, such as from ARF10 mRNA, accumulate in the xrn4 mutant, whereas others, such as from AGO1, do not. To examine the nature of this selectivity, transcripts that differentially accumulate in xrn4 were identified by combining PARE and Affymetrix arrays. Certain functional categories, such as stamen-associated proteins and hydrolases, were over-represented among transcripts decreased in xrn4, whereas transcripts encoding nuclear-encoded chloroplast-targeted proteins and nucleic acid-binding proteins were over-represented in transcripts increased in xrn4. To ascertain if RNA sequence influences the apparent XRN4 selectivity, a series of chimeric constructs was generated in which the miRNA-complementary sites and different portions of the surrounding sequences from AGO1 and ARF10 were interchanged. Analysis of the resulting transgenic plants revealed that the presence of a 150 nucleotide sequence downstream from the ARF10 miRNA-complementary site conferred strong accumulation of the 3' cleavage products in xrn4. In addition, sequence analysis of differentially accumulating transcripts led to the identification of 27 hexamer motifs that were over-represented in transcripts or miRNA-cleavage products accumulating in xrn4. Taken together, the data indicate that specific mRNA sequences, like those in ARF10, and mRNAs from select functional categories are attractive targets for XRN4-mediated decay.

Uridylation of mature miRNAs and siRNAs by the MUT68 nucleotidyltransferase promotes their degradation in Chlamydomonas.

Regulation of gene expression by small RNAs ( approximately 20-30 nucleotides in length) plays an essential role in developmental pathways and defense responses against genomic parasites in eukaryotes. MicroRNAs (miRNAs) and small interfering RNAs (siRNAs) commonly direct the inactivation of cognate sequences through a variety of mechanisms, including RNA degradation, translation inhibition, and transcriptional repression. Recent studies have provided considerable insight into the biogenesis and the mode of action of miRNAs and siRNAs. However, relatively little is known about mechanisms of quality control and small RNA decay in RNA interference (RNAi) pathways. Here we show that deletion of MUT68, encoding a terminal nucleotidyltransferase in the alga Chlamydomonas reinhardtii, results in elevated miRNA and siRNA levels. We found that MUT68 plays a role in the untemplated uridylation of the 3' ends of small RNAs in vivo and stimulates their degradation by the RRP6 exosome subunit in vitro. Moreover, RRP6 depletion also leads to accumulation of small RNAs in vivo. We propose that MUT68 and RRP6 cooperate in the degradation of mature miRNAs and siRNAs, as a quality control mechanism to eliminate dysfunctional or damaged small RNA molecules.

Diamonds in the rough: mRNA-like non-coding RNAs.

Non-coding RNAs are increasingly being identified as crucial regulators of gene expression and other cellular functions in plants. Experimental and computational methods have revealed the existence of mRNA-like non-coding RNAs (mlncRNAs), a class of non-coding RNAs that, in plants, are associated with tissue-specific expression, development and the phosphate-starvation response. Although their mechanisms of action are largely unknown, one can speculate that mlncRNAs act through secondary structures or specific sequences that bind to proteins or metabolites, or that have catalytic activity. This review summarizes the computational methods developed to identify candidate mlncRNAs, and the current experimental evidence regarding the function of several known mlncRNAs.

Sample sequencing of vascular plants demonstrates widespread conservation and divergence of microRNAs.

Small RNAs are pivotal regulators of gene expression that guide transcriptional and post-transcriptional silencing mechanisms in eukaryotes, including plants. Here we report a comprehensive atlas of sRNA and miRNA from 3 species of algae and 31 representative species across vascular plants, including non-model plants. We sequence and quantify sRNAs from 99 different tissues or treatments across species, resulting in a data set of over 132 million distinct sequences. Using miRBase mature sequences as a reference, we identify the miRNA sequences present in these libraries. We apply diverse profiling methods to examine critical sRNA and miRNA features, such as size distribution, tissue-specific regulation and sequence conservation between species, as well as to predict putative new miRNA sequences. We also develop database resources, computational analysis tools and a dedicated website, http://smallrna.udel.edu/. This study provides new insights on plant sRNAs and miRNAs, and a foundation for future studies.

Parallel analysis of RNA ends enhances global investigation of microRNAs and target RNAs of Brachypodium distachyon.

BACKGROUND: The wild grass Brachypodium distachyon has emerged as a model system for temperate grasses and biofuel plants. However, the global analysis of miRNAs, molecules known to be key for eukaryotic gene regulation, has been limited in B. distachyon to studies examining a few samples or that rely on computational predictions. Similarly an in-depth global analysis of miRNA-mediated target cleavage using parallel analysis of RNA ends (PARE) data is lacking in B. distachyon. RESULTS: B. distachyon small RNAs were cloned and deeply sequenced from 17 libraries that represent different tissues and stresses. Using a computational pipeline, we identified 116 miRNAs including not only conserved miRNAs that have not been reported in B. distachyon, but also non-conserved miRNAs that were not found in other plants. To investigate miRNA-mediated cleavage function, four PARE libraries were constructed from key tissues and sequenced to a total depth of approximately 70 million sequences. The roughly 5 million distinct genome-matched sequences that resulted represent an extensive dataset for analyzing small RNA-guided cleavage events. Analysis of the PARE and miRNA data provided experimental evidence for miRNA-mediated cleavage of 264 sites in predicted miRNA targets. In addition, PARE analysis revealed that differentially expressed miRNAs in the same family guide specific target RNA cleavage in a correspondingly tissue-preferential manner. CONCLUSIONS: B. distachyon miRNAs and target RNAs were experimentally identified and analyzed. Knowledge gained from this study should provide insights into the roles of miRNAs and the regulation of their targets in B. distachyon and related plants.

MAJOR DEVELOPMENTAL REGULATORS AND THEIR EXPRESSION IN TWO CLOSELY RELATED SPECIES OF PORPHYRA (RHODOPHYTA)(1).

Little is known about the genetic and biochemical mechanisms that underlie red algal development, for example, why the group failed to evolve complex parenchyma and tissue differentiation. Here we examined expressed sequence tag (EST) data from two closely related species, Porphyra umbilicalis (L.) J. Agardh and P. purpurea (Roth) C. Agardh, for conserved developmental regulators known from model eukaryotes, and their expression levels in several developmental stages. Genes for most major developmental families were present, including MADS-box and homeodomain (HD) proteins, SNF2 chromatin-remodelers, and proteins involved in sRNA biogenesis. Some of these genes displayed altered expression correlating with different life history stages or cell types. Notably, two ESTs encoding HD proteins showed eightfold higher expression in the P. purpurea sporophyte (conchocelis) than in the gametophyte (blade), whereas two MADS domain-containing paralogs showed significantly different patterns of expression in the conchocelis and blade respectively. These developmental gene families do not appear to have undergone the kinds of dramatic expansions in copy number found in multicellular land plants and animals, which are important for regulating developmental processes in those groups. Analyses of small RNAs did not validate the presence of miRNAs, but homologs of Argonaute were present. In general, it appears that red algae began with a similar molecular toolkit for directing development as did other multicellular eukaryotes, but probably evolved altered roles for many key proteins, as well as novel mechanisms yet to be discovered.

Abiotic stress-associated miRNAs: detection and functional analysis.

MicroRNAs (miRNAs) are small regulatory noncoding RNAs varying in length between 20 and 24 nucleotides. They play a key role during plant development by negatively regulating gene expression at the posttranscriptional level. Moreover, recent studies reported several miRNAs associated with abiotic stress responses. Small RNA cloning and high-throughput deep sequencing methods provide expression profiles of not only known miRNAs, but also novel miRNAs. In this chapter, we describe the methods used to identify and characterize abiotic stress-associated miRNAs and their target genes.

Global identification of microRNA-target RNA pairs by parallel analysis of RNA ends.

MicroRNAs (miRNAs) are important regulatory molecules in most eukaryotes and identification of their target mRNAs is essential for their functional analysis. Whereas conventional methods rely on computational prediction and subsequent experimental validation of target RNAs, we directly sequenced >28,000,000 signatures from the 5' ends of polyadenylated products of miRNA-mediated mRNA decay, isolated from inflorescence tissue of Arabidopsis thaliana, to discover novel miRNA-target RNA pairs. Within the set of approximately 27,000 transcripts included in the 8,000,000 nonredundant signatures, several previously predicted but nonvalidated targets of miRNAs were found. Like validated targets, most showed a single abundant signature at the miRNA cleavage site, particularly in libraries from a mutant deficient in the 5'-to-3' exonuclease AtXRN4. Although miRNAs in Arabidopsis have been extensively investigated, working in reverse from the cleaved targets resulted in the identification and validation of novel miRNAs. This versatile approach will affect the study of other aspects of RNA processing beyond miRNA-target RNA pairs.

The nucleus-encoded factor MCD4 participates in degradation of nonfunctional 3' UTR sequences generated by cleavage of pre-mRNA in Chlamydomonas chloroplasts.

The 3' maturation of chloroplast pre-mRNAs in Chlamydomonas proceeds via endonucleolytic cleavage, exonucleolytic trimming of the upstream cleavage product, and rapid degradation of the downstream moiety. However, the cis elements and trans factors remain to be characterized in detail. In the case of atpB, a 300 nucleotide processing determinant (PD), consisting of an inverted repeat (IR) and endonuclease cleavage site (ECS), directs 3' maturation. To further characterize the PD, 15 variants were examined in vivo in ectopic contexts. This revealed that the IR, and nucleotides 15-37 downstream of the ECS stimulate processing. A candidate trans factor for 3' maturation was subsequently functionally analyzed. This factor is encoded by the nuclear locus MCD4, and the mcd4 mutant was known to accumulate abnormally 3'-processed chloroplast mRNAs. When the mcd4 mutation was crossed into strains containing reporter genes with insertions of several PD versions, processing was reduced in some cases. This caused accumulation of RNA sequences downstream of the PD, which are normally degraded. From these data, it can be suggested that MCD4 facilitates the endonucleolytic cleavage step in 3' end maturation of atpB and perhaps other mRNAs, by interacting with the IR, RNA downstream of the IR, or with proteins bound there.

Nuclear suppressors define three factors that participate in both 5' and 3' end processing of mRNAs in Chlamydomonas chloroplasts.

Chloroplast RNA processing and degradation are orchestrated by nucleus-encoded factors. Although several transcript-specific factors have been identified, those involved in global RNA metabolism have mostly remained elusive. Using Chlamydomonas reinhardtii, we have identified three pleiotropic nuclear mutations, mcd3, mcd4 and mcd5, which cause quantitative variation between polycistronic transcripts and accumulation of transcripts with novel 3' ends. The mcd3, mcd4 and mcd5 mutants were initially isolated as photoautotrophic suppressors of the petD 5' mutants LS2 and LS6, which harbour four nucleotide linker-scanning mutations near the 5' end of the mature transcript. The LS mutants accumulate 1-3% of the wild-type (WT) petD mRNA level and no cytochrome b6/f complex subunit IV, which is the petD gene product and required for photosynthesis. Each suppressor restores approximately 15% of the WT petD mRNA and subunit IV levels. Genetic analysis showed mcd4 to be recessive, and suggested that MCD4 interacts with the petD mRNA stability factor MCD1. To assess the specificity of mcd3, mcd4 and mcd5, transcripts from 32 chloroplast genes were analysed by RNA filter hybridizations. mcd3 and mcd4 displayed aberrant transcript patterns for 17 genes, whereas only three were altered in mcd5. Since the mutations affect multiple RNAs in a variety of ways, our data suggest that MCD3, MCD4 and MCD5 may participate in a series of multiprotein complexes responsible for RNA maturation and degradation in Chlamydomonas chloroplasts.

Beyond complementation. Map-based cloning in Chlamydomonas reinhardtii.

Chlamydomonas reinhardtii is an excellent model system for plant biologists because of its ease of manipulation, facile genetics, and the ability to transform the nuclear, chloroplast, and mitochondrial genomes. Numerous forward genetics studies have been performed in Chlamydomonas, in many cases to elucidate the regulation of photosynthesis. One of the resultant challenges is moving from mutant phenotype to the gene mutation causing that phenotype. To date, complementation has been the primary method for gene cloning, but this is impractical in several situations, for example, when the complemented strain cannot be readily selected or in the case of recessive suppressors that restore photosynthesis. New tools, including a molecular map consisting of 506 markers and an 8X-draft nuclear genome sequence, are now available, making map-based cloning increasingly feasible. Here we discuss advances in map-based cloning developed using the strains mcd4 and mcd5, which carry recessive nuclear suppressors restoring photosynthesis to chloroplast mutants. Tools that have not been previously applied to Chlamydomonas, such as bulked segregant analysis and marker duplexing, are being implemented to increase the speed at which one can go from mutant phenotype to gene. In addition to assessing and applying current resources, we outline anticipated future developments in map-based cloning in the context of the newly extended Chlamydomonas genome initiative.