Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
Review
. 2018 Nov 9;4(4):31.
doi: 10.3390/ncrna4040031.

Contemporary Ribonomics Methods for Viral microRNA Target Analysis

Lauren A Gay  1 , Peter C Turner  2 , Rolf Renne  3
Affiliations
Review

Contemporary Ribonomics Methods for Viral microRNA Target Analysis

Lauren A Gay et al. Noncoding RNA. .

Abstract

Numerous cellular processes are regulated by microRNAs (miRNAs), both cellular and viral. Elucidating the targets of miRNAs has become an active area of research. An important method in this field is cross-linking and immunoprecipitation (CLIP), where cultured cells or tissues are UV-irradiated to cross-link protein and nucleic acid, the RNA binding protein of interest is immunoprecipitated, and the RNAs pulled down with the protein are isolated, reverse-transcribed, and analyzed by sequencing. CLIP using antibody against Argonaute (Ago), which binds to both miRNA and mRNA as they interact in RISC, has allowed researchers to uncover a large number of miRNA targets. Coupled with high-throughput sequencing, CLIP has been useful for revealing miRNA targetomes for the γ-herpesviruses Kaposi's sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV). Variants on the CLIP protocol are described, with the benefits and drawbacks of each. In particular, the most recent methods involving RNA⁻RNA ligation to join the miRNA and its RNA target have aided in target identification. Lastly, data supporting biologically meaningful interactions between miRNAs and long non-coding RNAs (lncRNAs) are reviewed. In summary, ribonomics-based miRNA targetome analysis has expanded our understanding of miRNA targeting and has provided a rich resource for EBV and KSHV research with respect to pathogenesis and tumorigenesis.

Keywords: Ago; CLASH; CLIP; EBV; HITS-CLIP; KSHV; PAR-CLIP; microRNA; qCLASH.

PubMed Disclaimer

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Outline of high-throughput sequencing cross-linking and immunoprecipitation (HITS-CLIP), photoactivatable-ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) and cross-linking and sequencing of hybrids (CLASH) ribonomics protocols, with significant differences indicated. The steps from UV irradiation of cells through to sequencing library construction are shown. 4SU: 4-thiouridine; Ago: Argonaute. The black circles indicate mutations introduced by cross-linking damage, and the squares the nucleotide incorporated following reverse transcription. For PAR-CLIP, reverse transcription of RNA containing a crosslinked 4SU results in the misincorporation of a G in the opposite strand instead of A. The 3′ and 5′ adaptors are shown in red, and in orange following conversion to cDNA. Reproduced with permission from Sethuraman et al., Nucleic Acids Research; published by Oxford University Press, 2018, [12].
Figure 2
Figure 2
(A) The quick CLASH (qCLASH) method. The shaded box indicates the portion of the method that takes place on beads; (B) The two possible ways in which microRNAs (miRNAs) and mRNAs can join during intermolecular ligation. From [27], Copyright © 2018 American Society for Microbiology, Journal of Virology, 92, e02138-17, 2018.
Figure 3
Figure 3
Patterns of binding for selected individual Kaposi’s sarcoma-associated herpesvirus (KSHV) miRNAs. The status of each nucleotide (bound or unbound) along the length of the miRNA portion of each hybrid was determined, based on the Vienna diagrams generated through Hyb. BR1, BR2 and BR3 indicates biological replicates 1, 2 and 3. (A) KSHV miR-K12-1; (B) KSHV miR-K12-6-5p; (C) KSHV miR-K12-3. Adapted from [27], Copyright © 2018 American Society for Microbiology, Journal of Virology, 92, e02138-17, 2018.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

References

    1. Nelson P., Kiriakidou M., Sharma A., Maniataki E., Mourelatos Z. The microRNA world: Small is mighty. Trends Biochem. Sci. 2003;28:534–540. doi: 10.1016/j.tibs.2003.08.005. - DOI - PubMed
    1. Bartel D.P. MicroRNAs: Genomics, biogenesis, mechanism, and function. Cell. 2004;116:281–297. doi: 10.1016/S0092-8674(04)00045-5. - DOI - PubMed
    1. Hock J., Meister G. The Argonaute protein family. Genome Biol. 2008;9:210. doi: 10.1186/gb-2008-9-2-210. - DOI - PMC - PubMed
    1. Bernier A., Sagan S.M. The diverse roles of microRNAs at the host(-)virus interface. Viruses. 2018;10:440. doi: 10.3390/v10080440. - DOI - PMC - PubMed
    1. Vojtechova Z., Tachezy R. The role of miRNAs in virus-mediated oncogenesis. Int. J. Mol. Sci. 2018;19:1217. doi: 10.3390/ijms19041217. - DOI - PMC - PubMed

LinkOut - more resources