MicroRNAs: Genomics, Biogenesis, Mechanism, and Function

RNA interference (RNAi) is the process of sequence-specific, post-transcriptional gene silencing in animals and plants, initiated by double-stranded RNA (dsRNA) that is homologous in sequence to the silenced gene. The mediators of sequence-specific messenger RNA degradation are 21- and 22-nucleotide small interfering RNAs (siRNAs) generated by ribonuclease III cleavage from longer dsRNAs. Here we show that 21-nucleotide siRNA duplexes specifically suppress expression of endogenous and heterologous genes in different mammalian cell lines, including human embryonic kidney (293) and HeLa cells. Therefore, 21-nucleotide siRNA duplexes provide a new tool for studying gene function in mammalian cells and may eventually be used as gene-specific therapeutics.

Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells

MicroRNAs are a family of small, non-coding RNAs that regulate gene expression in a sequence-specific manner. The two founding members of the microRNA family were originally identified in Caenorhabditis elegans as genes that were required for the timed regulation of developmental events. Since then, hundreds of microRNAs have been identified in almost all metazoan genomes, including worms, flies, plants and mammals. MicroRNAs have diverse expression patterns and might regulate various developmental and physiological processes. Their discovery adds a new dimension to our understanding of complex gene regulatory networks.

/pdf/micrornas-small-rnas-with-a-big-role-in-gene-regulation-3a05xihg95.pdf

MicroRNAs: small RNAs with a big role in gene regulation

The genomes of many animals, plants and fungi are tagged by methylation of DNA cytosine. To understand the biological significance of this epigenetic mark it is essential to know where in the genome it is located. New techniques are making it easier to map DNA methylation patterns on a large scale and the results have already provided surprises. In particular, the conventional view that DNA methylation functions predominantly to irreversibly silence transcription is being challenged. Not only is promoter methylation often highly dynamic during development, but many organisms also seem to target DNA methylation specifically to the bodies of active genes.

https://birdlab.bio.ed.ac.uk/bird/sites/sbsweb2.bio.ed.ac.uk.bird/files/20.pdf

DNA methylation landscapes: provocative insights from epigenomics

Gene silencing through RNA interference (RNAi) is carried out by RISC, the RNA-induced silencing complex. RISC contains two signature components, small interfering RNAs (siRNAs) and Argonaute family proteins. Here, we show that the multiple Argonaute proteins present in mammals are both biologically and biochemically distinct, with a single mammalian family member, Argonaute2, being responsible for messenger RNA cleavage activity. This protein is essential for mouse development, and cells lacking Argonaute2 are unable to mount an experimental response to siRNAs. Mutations within a cryptic ribonuclease H domain within Argonaute2, as identified by comparison with the structure of an archeal Argonaute protein, inactivate RISC. Thus, our evidence supports a model in which Argonaute contributes "Slicer" activity to RISC, providing the catalytic engine for RNAi.

https://science.sciencemag.org/content/sci/305/5689/1437.full.pdf

Argonaute2 Is the Catalytic Engine of Mammalian RNAi

Double‐stranded RNA induces a post‐transcriptional gene silencing process, termed RNAi, in diverse organisms. It is shown here that transcriptional gene silencing accompanied by de novo methylation of a target promoter in plants can be triggered by a double‐stranded RNA containing promoter sequences. Similar to the double‐stranded RNA involved in RNAi, this promoter double‐stranded RNA, which is synthesized in the nucleus, is partially cleaved into small RNAs ∼23 nucleotides in length. Both transcriptional and post‐transcriptional gene silencing can thus be initiated by double‐stranded RNAs that enter the same degradation pathway. The results also implicate double‐stranded RNA in directing DNA methylation. Different constructs designed to produce double‐stranded promoter RNA in various ways were evaluated for their ability to induce gene silencing in tobacco and Arabidopsis . RNA hairpins transcribed from inverted DNA repeats were the most effective trans ‐acting silencing signals. This strategy could be useful for transcriptionally downregulating genes in a variety of plants.

/pdf/transcriptional-silencing-and-promoter-methylation-triggered-x2zaghjds1.pdf

Transcriptional silencing and promoter methylation triggered by double-stranded RNA

Previous work has suggested that de novo methylation of plant nuclear genes can be triggered by an RNA–DNA interaction. To test whether transcription of a promoter would induce de novo methylation and silencing of unlinked genes driven by the same promoter, a chimeric ‘gene’ consisting of a nopaline synthase promoter (NOSpro) positioned downstream of the cauliflower mosaic virus 35S promoter (35Spro) and flanked at the 3′ end by a NOS terminator (NOSter) was constructed and introduced into the genome of a plant that normally expresses an unmethylated NOSpro‐neomycinphosphotransferase ( nptII ) gene. Transformants were tested for kanamycin resistance and NOSpro RNA synthesis. Most produced a full‐length polyadenylated NOSpro RNA, which did not induce silencing or methylation at the NOSpro‐ nptII target gene. One, however, contained truncated non‐polyadenylated NOSpro RNA; in this plant, the NOSpro‐ nptII gene became silenced and methylated in the NOSpro region. Molecular analysis of the NOSpro silencing locus revealed two incomplete copies of the 35Spro‐NOSpro gene arranged as an inverted repeat with NOSpro sequences at the center. Reducing NOSpro transcription by crossing a 35Spro‐silencing locus partially reactivated nptII gene expression and decreased NOSpro methylation at the target locus, thus implicating aberrant NOSpro RNA in this trans ‐silencing phenomenon.

/pdf/production-of-aberrant-promoter-transcripts-contributes-to-12sxjcs5al.pdf

Production of aberrant promoter transcripts contributes to methylation and silencing of unlinked homologous promoters in trans

Although integration of viral DNA into host chromosomes occurs regularly in bacteria and animals, there are few reported cases in plants, and these involve insertion at only one or a few sites. Here, we report that pararetrovirus-like sequences have integrated repeatedly into tobacco chromosomes, attaining a copy number of ≈103. Insertion apparently occurred by illegitimate recombination. From the sequences of 22 independent insertions recovered from a healthy plant, an 8-kilobase genome encoding a previously uncharacterized pararetrovirus that does not contain an integrase function could be assembled. Preferred boundaries of the viral inserts may correspond to recombinogenic gaps in open circular viral DNA. An unusual feature of the integrated viral sequences is a variable tandem repeat cluster, which might reflect defective genomes that preferentially recombine into plant DNA. The recurrent invasion of pararetroviral DNA into tobacco chromosomes demonstrates that viral sequences can contribute significantly to plant genome evolution.

https://www.pnas.org/content/pnas/96/23/13241.full.pdf

Integrated pararetroviral sequences define a unique class of dispersed repetitive DNA in plants.

Tobacco endogenous pararetroviruses (TEPRVs) represent the first virus-derived repetitive sequence family found in plants. The sequence conservation of TEPRVs and the lack of an exogenous form of the virus suggest that TEPRVs serve a beneficial function, perhaps by furnishing virus resistance via homologous sequence interactions. This hypothesis is supported by the observation that TEPRVs are methylated and negligibly transcribed. Moreover, transgenes driven by the TEPRV enhancer are silenced and methylated when introduced into tobacco, but remain active and unmethylated in non-host species devoid of sequences homologous to TEPRVs. In transgenic Arabidopsis, the TEPRV enhancer is active primarily in shoot meristems. This suggests that the virus giving rise to TEPRVs could infect germ cell precursors, a prerequisite for meiotically heritable insertions into host chromosomes. The copy number, organization and methylation of TEPRVs in tetraploid tobacco and one of its diploid ancestors, Nicotiana sylvestris, the presumed original host for the virus, have remained constant since polyploid formation. The remarkable conservation of these features in two independently evolving species further supports a role for TEPRVs in viral immunity.

/pdf/endogenous-viral-sequences-and-their-potential-contribution-3kk49qjwna.pdf

J. van der Winden

Papers

Transcriptional silencing and promoter methylation triggered by double-stranded RNA

Production of aberrant promoter transcripts contributes to methylation and silencing of unlinked homologous promoters in trans

Integrated pararetroviral sequences define a unique class of dispersed repetitive DNA in plants.

Endogenous viral sequences and their potential contribution to heritable virus resistance in plants.