Sequence-non-specific effects of RNA interference triggers and microRNA regulators

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Abstract

RNA reagents of diverse lengths and structures, unmodified or containing various chemical modifications are powerful tools of RNA interference and microRNA technologies. These reagents which are either delivered to cells using appropriate carriers or are expressed in cells from suitable vectors often cause unintended sequence-non-specific immune responses besides triggering intended sequence-specific silencing effects. This article reviews the present state of knowledge regarding the cellular sensors of foreign RNA, the signaling pathways these sensors mobilize and shows which specific features of the RNA reagents set the responsive systems on alert. The representative examples of toxic effects caused in the investigated cell lines and tissues by the RNAs of specific types and structures are collected and may be instructive for further studies of sequence-non-specific responses to foreign RNA in human cells.

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Most cited references 159

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Microarray analysis shows that some microRNAs downregulate large numbers of target mRNAs.

Lee P. Lim, Nelson C. Lau, Philip Garrett-Engele … (2005)

MicroRNAs (miRNAs) are a class of noncoding RNAs that post-transcriptionally regulate gene expression in plants and animals. To investigate the influence of miRNAs on transcript levels, we transfected miRNAs into human cells and used microarrays to examine changes in the messenger RNA profile. Here we show that delivering miR-124 causes the expression profile to shift towards that of brain, the organ in which miR-124 is preferentially expressed, whereas delivering miR-1 shifts the profile towards that of muscle, where miR-1 is preferentially expressed. In each case, about 100 messages were downregulated after 12 h. The 3' untranslated regions of these messages had a significant propensity to pair to the 5' region of the miRNA, as expected if many of these messages are the direct targets of the miRNAs. Our results suggest that metazoan miRNAs can reduce the levels of many of their target transcripts, not just the amount of protein deriving from these transcripts. Moreover, miR-1 and miR-124, and presumably other tissue-specific miRNAs, seem to downregulate a far greater number of targets than previously appreciated, thereby helping to define tissue-specific gene expression in humans.

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Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics.

Shao-En Ong, Blagoy Blagoev, Irina Kratchmarova … (2002)

Quantitative proteomics has traditionally been performed by two-dimensional gel electrophoresis, but recently, mass spectrometric methods based on stable isotope quantitation have shown great promise for the simultaneous and automated identification and quantitation of complex protein mixtures. Here we describe a method, termed SILAC, for stable isotope labeling by amino acids in cell culture, for the in vivo incorporation of specific amino acids into all mammalian proteins. Mammalian cell lines are grown in media lacking a standard essential amino acid but supplemented with a non-radioactive, isotopically labeled form of that amino acid, in this case deuterated leucine (Leu-d3). We find that growth of cells maintained in these media is no different from growth in normal media as evidenced by cell morphology, doubling time, and ability to differentiate. Complete incorporation of Leu-d3 occurred after five doublings in the cell lines and proteins studied. Protein populations from experimental and control samples are mixed directly after harvesting, and mass spectrometric identification is straightforward as every leucine-containing peptide incorporates either all normal leucine or all Leu-d3. We have applied this technique to the relative quantitation of changes in protein expression during the process of muscle cell differentiation. Proteins that were found to be up-regulated during this process include glyceraldehyde-3-phosphate dehydrogenase, fibronectin, and pyruvate kinase M2. SILAC is a simple, inexpensive, and accurate procedure that can be used as a quantitative proteomic approach in any cell culture system.

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An abundant class of tiny RNAs with probable regulatory roles in Caenorhabditis elegans.

N. Lau, L. P. Lim, E G Weinstein … (2001)

Two small temporal RNAs (stRNAs), lin-4 and let-7, control developmental timing in Caenorhabditis elegans. We find that these two regulatory RNAs are members of a large class of 21- to 24-nucleotide noncoding RNAs, called microRNAs (miRNAs). We report on 55 previously unknown miRNAs in C. elegans. The miRNAs have diverse expression patterns during development: a let-7 paralog is temporally coexpressed with let-7; miRNAs encoded in a single genomic cluster are coexpressed during embryogenesis; and still other miRNAs are expressed constitutively throughout development. Potential orthologs of several of these miRNA genes were identified in Drosophila and human genomes. The abundance of these tiny RNAs, their expression patterns, and their evolutionary conservation imply that, as a class, miRNAs have broad regulatory functions in animals.

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Author and article information

Journal

Journal ID (nlm-ta): Nucleic Acids Res

Journal ID (iso-abbrev): Nucleic Acids Res

Journal ID (publisher-id): nar

Journal ID (hwp): nar

Title: Nucleic Acids Research

Publisher: Oxford University Press

ISSN (Print): 0305-1048

ISSN (Electronic): 1362-4962

Publication date Collection: January 2010

Publication date (Print): January 2010

Publication date (Electronic): 20 October 2009

Publication date PMC-release: 20 October 2009

Volume: 38

Issue: 1

Pages: 1-16

Affiliations

Laboratory of Cancer Genetics, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego 12/14, 61-704 Poznan, Poland

Author notes

*To whom correspondence should be addressed. Tel: +48 618528503; Fax: +48 618520532; Email: wlodkrzy@ 123456ibch.poznan.pl

Article

Publisher ID: gkp829

DOI: 10.1093/nar/gkp829

PMC ID: 2800214

PubMed ID: 19843612

SO-VID: 19345c43-ea43-49e1-9e16-ca3f8718f6ce

License:

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( http://creativecommons.org/licenses/by-nc/2.5/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

History

Date received : 24 July 2009

Date revision received : 1 September 2009

Date accepted : 20 September 2009

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Sequence-non-specific effects of RNA interference triggers and microRNA regulators

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Abstract

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RNA drug delivery

Most cited references 159

Microarray analysis shows that some microRNAs downregulate large numbers of target mRNAs.

Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics.

An abundant class of tiny RNAs with probable regulatory roles in Caenorhabditis elegans.

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Cited by 128

Most referenced authors 3,862