Identification and characterization of small RNAs involved in RNA silencing

Aravin, Alexei A.; Tuschl, Thomas

doi:10.1016/j.febslet.2005.08.009

Cited by 210 publications

(160 citation statements)

References 116 publications

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“…Are rasiRNAs generated and are they involved in a similar RNAi-mediated silencing mechanism in mammalian cells (see reviews on RNAi-mediated heterochromatic gene silencing 86,90,91 )? Numerous rasiRNAs have been recently identified in mouse eggs and early embryos, which shows that fold-back dsRNAs of mammalian retrotransposon sequences can be processed to rasiRNAs 92 .…”

Section: Suppression Of Rasirna?mentioning

confidence: 99%

“…rasiRNAs are proposed to constrain the expression of retro elements and protect the genome integrity of eggs and early embryos by the RNAi-mediated heterochromatic silencing mechanism 86,88,89 . The details of how rasiRNAs activate the mechanism are unknown.Are rasiRNAs generated and are they involved in a similar RNAi-mediated silencing mechanism in mammalian cells (see reviews on RNAi-mediated heterochromatic gene silencing 86,90,91 )? Numerous rasiRNAs have been recently identified in mouse eggs and early embryos, which shows that fold-back dsRNAs of mammalian retrotransposon sequences can be processed to rasiRNAs 92 .…”

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confidence: 99%

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Editor meets silencer: crosstalk between RNA editing and RNA interference

Nishikura

2006

Nat Rev Mol Cell Biol

257

263

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The most prevalent type of RNA editing is mediated by ADAR (adenosine deaminase acting on RNA) enzymes, which convert adenosines to inosines (a process known as A→I RNA editing) in double-stranded (ds)RNA substrates. A→I RNA editing was long thought to affect only selected transcripts by altering the proteins they encode. However, genome-wide screening has revealed numerous editing sites within inverted Alu repeats in introns and untranslated regions. Also, recent evidence indicates that A→I RNA editing crosstalks with RNA-interference pathways, which, like A→I RNA editing, involve dsRNAs. A→I RNA editing therefore seems to have additional functions, including the regulation of retrotransposons and gene silencing, which adds a new urgency to the challenges of fully understanding ADAR functions.An RNA transcript is subjected to various maturation processes, such as 5′ capping, splicing, 3′ processing and polyadenylation, after it is transcribed from the gene. Post-transcriptional processing of primary transcripts is essential to generate mature messenger RNAs that are ready to be translated into proteins 1 . RNA editing is a post-transcriptional-processing mechanism that results in an RNA sequence that is different from the one encoded by the genome, and thereby contributes to the diversity of gene products. There are different types of RNA-editing mechanism that either add or delete nucleotides, or that change one nucleotide into another 2 (BOX 1).The type of RNA editing that is most prevalent in higher eukaryotes converts adenosine (A) residues into inosine (I) in double-stranded (ds)RNAs through the action of ADAR (adenosine deaminase acting on RNA) enzymes [3][4][5] . A→I RNA editing of a short dsRNA that has formed between a coding exon and nearby intron sequences can lead to a codon change and an alteration in the protein function. However, it was recently discovered that the most frequent targets of A→I RNA editing seem to be long, but partially double-stranded, RNAs that are formed from inverted Alu repeats and long interspersed element (LINE) repeats located in introns and untranslated regions (UTRs) of mRNAs [6][7][8][9] . Global editing of non-coding RNA might control the expression of genes that harbour these repeat sequences of retrotransposon origin.Post-transcriptional gene regulation can also occur through RNA interference (RNAi), an evolutionarily conserved phenomenon that involves dsRNA molecules 10,11 . Small interfering RNAs (siRNAs) and microRNAs (miRNAs) are non-coding RNAs that are generated by a class of RNase III ribonucleases (specifically, Dicer and Drosha). These small RNAs are incorporated into the RNA-induced silencing complex (RISC), which mediates the RNAi process [12][13][14][15][16] . The idea that the RNAi and A→I RNA editing pathways might compete for a Competing interests statement: The author declares no competing financial interests. [23][24][25] . NIH Public AccessIn this review, I discuss recent findings on new functions of A→I RNA editing in the regulation of non...

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Section: Suppression Of Rasirna?mentioning

confidence: 99%

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confidence: 99%

Editor meets silencer: crosstalk between RNA editing and RNA interference

Nishikura

2006

Nat Rev Mol Cell Biol

257

263

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“…They have been shown to play an important role in many different cellular, developmental, and physiological processes as divergent as cell lineage decisions, cell proliferation, apoptosis, morphogenesis, fat metabolism, hormone secretion, neuronal synaptic plasticity, and long-term memory (Aravin and Tuschl 2005). Methods that have been established to identify and quantify miRNAs include Northern blotting (Lim et al 2003), in situ hybridization (Pena et al 2009), small RNA library sequencing (Lagos-Quintana et al 2001), bead arrays (Chen et al 2008), microarray hybridization (Krichevsky et al 2003;Barad et al 2004;Liu et al 2004;Landgraf et al 2007), and reverse transcription PCR (Chen et al 2005).…”

Section: Introductionmentioning

confidence: 99%

Absolute quantification of microRNAs by using a universal reference

Bissels¹,

Wild²,

Tomiuk³

et al. 2009

RNA

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MicroRNAs (miRNAs) are a species of small RNAs ;21-23-nucleotides long that have been shown to play an important role in many different cellular, developmental, and physiological processes. Accordingly, numerous PCR-, sequencing-, or hybridization-based methods have been established to identify and quantify miRNAs. Their short length results in a high dynamic range of melting temperatures and therefore impedes a proper selection of detection probes or optimized PCR primers. While miRNA microarrays allow for massive parallel and accurate relative measurement of all known miRNAs, they have so far been less useful as an assay for absolute quantification. Here, we present a microarray-based approach for global and absolute quantification of miRNAs. The method relies on the parallel hybridization of the sample of interest labeled with Cy5 and a universal reference of 954 synthetic miRNAs in equimolar concentrations that are labeled with Cy3 on a microarray slide containing probes for all human, mouse, rat, and viral miRNAs (miRBase 12.0). Each single miRNA is quantified with respect to the universal reference canceling biases related to sequence, labeling, or hybridization. We demonstrate the accuracy of the method by various spike-in experiments. Furthermore, we quantified miRNA copy numbers in liver samples and CD34(+)/ CD133(À) hematopoietic progenitor cells.

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“…The AGPC method, meanwhile, allows the recovery of all types of RNA molecules, including small RNAs in relatively cheap price than column-based commercial small RNA extraction kits [19,20]. Nonetheless, in order to purify small RNA based on AGPC method, extra purification step is still necessary to separate only small RNA portions from the total RNAs and it require large amounts of total RNAs in the initial stage of the sample preparation.…”

Section: Introductionmentioning

confidence: 99%

mirRICH, a simple method to enrich the small RNA fraction from over-dried RNA pellets

et al. 2018

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Techniques to isolate the small RNA fraction (<200nt) by column-based methods are commercially available. However, their use is limited because of the relatively high cost. We found that large RNA molecules, including mRNAs and rRNAs, are aggregated together in the presence of salts when RNA pellets are over-dried. Moreover, once RNA pellets are over-dried, large RNA molecules are barely soluble again during the elution process, whereas small RNA molecules (<100nt) can be eluted. We therefore modified the acid guanidinium thiocyanate-phenol-chloroform (AGPC)-based RNA extraction protocol by skipping the 70% ethanol washing step and over-drying the RNA pellet for 1 hour at room temperature. We named this novel small RNA isolation method “mirRICH.” The quality of the small RNA sequences was validated by electrophoresis, next-generation sequencing, and quantitative PCR, and the findings support that our newly developed column-free method can successfully and efficiently isolate small RNAs from over-dried RNA pellets.

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Identification and characterization of small RNAs involved in RNA silencing

Cited by 210 publications

References 116 publications

Editor meets silencer: crosstalk between RNA editing and RNA interference

Editor meets silencer: crosstalk between RNA editing and RNA interference

Absolute quantification of microRNAs by using a universal reference

mirRICH, a simple method to enrich the small RNA fraction from over-dried RNA pellets

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