Components Of Small RNA Pathways Research Articles

The alg-1 Gene Is Necessary for Orsay Virus Replication in Caenorhabditis elegans.

The establishment of the Orsay virus-Caenorhabditis elegans infection model has enabled the identification of host factors essential for virus infection. Argonautes are RNA interacting proteins evolutionary conserved in the three domains of life that are key components of small RNA pathways. C. elegans encodes 27 argonautes or argonaute-like proteins. Here, we determined that mutation of the argonaute-like gene 1, alg-1, results in a greater than 10,000-fold reduction in Orsay viral RNA levels, which could be rescued by ectopic expression of alg-1. Mutation in ain-1, a known interactor of ALG-1 and component of the RNA-induced silencing complex, also resulted in a significant reduction in Orsay virus levels. Viral RNA replication from an endogenous transgene replicon system was impaired by the lack of ALG-1, suggesting that ALG-1 plays a role during the replication stage of the virus life cycle. Orsay virus RNA levels were unaffected by mutations in the ALG-1 RNase H-like motif that ablate the slicer activity of ALG-1. These findings demonstrate a novel function of ALG-1 in promoting Orsay virus replication in C. elegans. IMPORTANCE All viruses are obligate intracellular parasites that recruit the cellular machinery of the host they infect to support their own proliferation. We used Caenorhabditis elegans and its only known infecting virus, Orsay virus, to identify host proteins relevant for virus infection. We determined that ALG-1, a protein previously known to be important in influencing worm life span and the expression levels of thousands of genes, is required for Orsay virus infection of C. elegans. This is a new function attributed to ALG-1 that was not recognized before. In humans, it has been shown that AGO2, a close relative protein to ALG-1, is essential for hepatitis C virus replication. This demonstrates that through evolution from worms to humans, some proteins have maintained similar functions, and consequently, this suggests that studying virus infection in a simple worm model has the potential to provide novel insights into strategies used by viruses to proliferate.

Contribution of Small RNA Pathway Components in Plant Immunity

Small RNAs regulate a multitude of cellular processes, including development, stress responses, metabolism, and maintenance of genome integrity, in a sequence-specific manner. Accumulating evidence reveals that host endogenous small RNAs and small RNA pathway components play important roles in plant immune responses against various pathogens, including bacteria, fungi, oomycetes, and viruses. Small-RNA-mediated defense responses are regulated through diverse pathways and the components of these pathways, including Dicer-like proteins, RNA-dependent RNA polymerases, Argonaute proteins, and RNA polymerase IV and V, exhibit functional specificities as well as redundancy. In this review, we summarize the recent insights revealed mainly through the examination of two model plants, Arabidopsis and rice, with a primary focus on our emerging understanding of how these small RNA pathway components contribute to plant immunity.

In Medicago truncatula, water deficit modulates the transcript accumulation of components of small RNA pathways.

BackgroundSmall RNAs (sRNAs) are 20-24 nucleotide (nt) RNAs and are involved in plant development and response to abiotic stresses. Plants have several sRNA pathways implicated in the transcriptional and post-transcriptional silencing of gene expression. Two key enzyme families common to all pathways are the Dicer-like (DCL) proteins involved in sRNAs maturation and the Argonautes (AGOs) involved in the targeting and functional action of sRNAs. Post-transcriptional silencing mediated by AGOs may occur by cleavage or translational repression of target mRNA's, while transcriptional silencing may be controlled by DNA methylation and chromatin remodeling. Thus far, these gene families have not been characterized in legumes, nor has their involvement in adaptation to water deficit been studied.ResultsA bioinformatic search in Medicago truncatula genome databases, using Arabidopsis thaliana AGO and DCL cDNA and protein sequences, identified three sequences encoding for putative Dicer-like genes and twelve sequences encoding for putative Argonaute genes. Under water deficit conditions and mainly in roots, MtDCL1 and MtAGO1, two enzymes probably involved in the processing and activation of microRNAs (miRNAs), increased their transcript levels. mir162 which target DCL1 mRNA and mir168 which target AGO1 mRNA reduced their expression in the roots of plants subjected to water deficit. Three putative genes, MtDCL3, MtAGO4b and MtAGO4c probably involved in DNA methylation mechanisms, increased their mRNA levels. However, the mRNA levels of MtAGO6 reduced, which probably encodes a protein with functions similar to MtAGO4. MtAGO7 mRNA levels increased and possibly encodes a protein involved in the production of trans-acting small interfering RNAs. The transcript abundance of MtAGO12a, MtAGO12b and MtAGO12c reduced under water deprivation. Plants recovered from water deprivation reacquire the mRNA levels of the controls.ConclusionsOur work demonstrates that in M. truncatula the transcript accumulation of the components of small RNA pathways is being modulated under water deficit. This shows that the transcriptional and post-transcriptional control of gene expression mediated by sRNAs is probably involved in plant adaptation to abiotic environmental changes. In the future this will allow the manipulation of these pathways providing a more efficient response of legumes towards water shortage.

Role of Small RNAs in Host-Microbe Interactions

Plant defense responses against pathogens are mediated by activation and repression of a large array of genes. Host endogenous small RNAs are essential in this gene expression reprogramming process. Here, we discuss recent findings on pathogen-regulated host microRNAs (miRNAs) and small interfering RNAs (siRNAs) and their roles in plant-microbe interaction. We further introduce small RNA pathway components, including Dicer-like proteins (DCLs), double-stranded RNA (dsRNA) binding protein, RNA-dependent RNA polymerases (RDRs), small RNA methyltransferase HEN1, and Argonaute (AGO) proteins, that contribute to plant immune responses. The strategies that pathogens have evolved to suppress host small RNA pathways are also discussed. Collectively, host small RNAs and RNA silencing machinery constitute a critical layer of defense in regulating the interaction of pathogens with plants.

Small RNA Pathways Are Present and Functional in the Angiosperm Male Gametophyte

Small non-coding RNAs are essential for development of the sporophyte, the somatic diploid phase of flowering plants. They are integral to key cellular processes such as defense, generation of chromatin structure, and regulation of native gene expression. Surprisingly, very little is known of their presence and function in the male haploid phase of plant development (male gametophyte/pollen grain), where dramatic cell fate changes leading to gametogenesis occur over just two mitotic divisions. We show that critical components of small RNA pathways are expressed throughout pollen development, but in a pattern that differs from the sporophyte. We also demonstrate that mature pollen accumulates a range of mature microRNAs, the class of small RNA most frequently involved in post-transcriptional regulation of endogenous gene expression. Significantly, these miRNAs cleave their target transcripts in developing pollen-a process that seemingly contributes to the purging of key regulatory transcripts from the mature pollen grain. Small RNAs are thus likely to make a hitherto unappreciated contribution to male gametophyte gene expression patterns, pollen development, and gametogenesis.

Small RNA-directed heterochromatin formation in the context of development: What flies might learn from fission yeast

A link between the RNAi system and heterochromatin formation has been established in several model organisms including Schizosaccharomyces pombe and Arabidopsis thaliana. However, the data to support a role for small RNAs and the associated machinery in transcriptional gene silencing in animal systems is more tenuous. Using the S. pombe system as a model, we analyze the role of small RNA pathway components and associated small RNAs in regulating transposable elements and potentially directing heterochromatin formation at these elements in Drosophila melanogaster.

Gene Family Analysis of the Arabidopsis Pollen Transcriptome Reveals Biological Implications for Cell Growth, Division Control, and Gene Expression Regulation

Upon germination, pollen forms a tube that elongates dramatically through female tissues to reach and fertilize ovules. While essential for the life cycle of higher plants, the genetic basis underlying most of the process is not well understood. We previously used a combination of flow cytometry sorting of viable hydrated pollen grains and GeneChip array analysis of one-third of the Arabidopsis (Arabidopsis thaliana) genome to define a first overview of the pollen transcriptome. We now extend that study to approximately 80% of the genome of Arabidopsis by using Affymetrix Arabidopsis ATH1 arrays and perform comparative analysis of gene family and gene ontology representation in the transcriptome of pollen and vegetative tissues. Pollen grains have a smaller and overall unique transcriptome (6,587 genes expressed) with greater proportions of selectively expressed (11%) and enriched (26%) genes than any vegetative tissue. Relative gene ontology category representations in pollen and vegetative tissues reveal a functional skew of the pollen transcriptome toward signaling, vesicle transport, and the cytoskeleton, suggestive of a commitment to germination and tube growth. Cell cycle analysis reveals an accumulation of G2/M-associated factors that may play a role in the first mitotic division of the zygote. Despite the relative underrepresentation of transcription-associated transcripts, nonclassical MADS box genes emerge as a class with putative unique roles in pollen. The singularity of gene expression control in mature pollen grains is further highlighted by the apparent absence of small RNA pathway components.