HITS-CLIP and PAR-CLIP advance viral miRNA targetome analysis

Abstract

MiRNAs regulate gene expression by binding predominantly to the 3' untranslated region (UTR) of target transcripts to prevent their translation and/or induce target degradation. In addition to the more than 1200 human miRNAs, human DNA tumor viruses such as Kaposi's sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV) encode miRNAs. Target predictions indicate that each miRNA targets hundreds of transcripts, many of which are regulated by multiple miRNAs. Thus, target identification is a big challenge for the field. Most methods used currently investigate single miRNA-target interactions and are not able to analyze complex miRNA-target networks. To overcome these challenges, cross-linking and immunoprecipitation (CLIP), a recently developed method to study direct RNA-protein interactions in living cells, has been successfully applied to miRNA target analysis. It utilizes Argonaute (Ago)-immunoprecipitation to isolate native Ago-miRNA-mRNA complexes. In four recent publications, two variants of the CLIP method (HITS-CLIP and PAR-CLIP) were utilized to determine the targetomes of human and viral miRNAs in cells infected with the gamma-herpesviruses KSHV and EBV, which are associated with a number of human cancers. Here, we briefly introduce herpesvirus-encoded miRNAs and then focus on how CLIP technology has largely impacted our understanding of viral miRNAs in viral biology and pathogenesis.

Figure 1. Comparison of HITS-CLIP and PAR-CLIP experimental procedures

RNA-protein cross-linking occurs in living cells. HITS-CLIP uses cross-linking at 254 nm, while in PAR-CLIP cells are fed with 4-Thiouridine (4SU), which is incorporated into nascent RNA and cross-links at 365 nm. Cross-linked cells are lysed and lysates are treated with RNase to trim the mRNA. In HITS-CLIP mRNAs are partially digested with RNase A, which cleaves after pyrimidines, leaving mRNAs with lengths between 50 and >100 bp. PAR-CLIP performs an extensive RNA digest with RNase T1, which cleaves after guanine nucleotides, resulting in very short mRNA fragments of 20-30 nt. After Ago-immunoprecipitation, linker ligation and radioactive labeling of the RNA, cross-linked Ago-miRNA-mRNA complexes are further purified by SDS-PAGE. HITS-CLIP yields two major bands, the Ago-miRNA complexes migrating at ∼100 kDa, and the Ago-mRNA and double cross-linked Ago-miRNA-mRNA complexes migrating at 120-130 kDa (corresponding to varying mRNA lengths between 50- and 80 bp). PAR-CLIP produces only one band at 100-110 kDa containing both RNA species. Complexes are cut out and RNAs are extracted and ligated to the 5′ linker. Reverse transcription and PCR amplification creates the final libraries for Illumina sequencing. In PAR-CLIP, the cross-linked 4SU base pairs with Guanosine instead of Adenosine during reverse transcription, resulting in a T to C transition in the final PCR product that allows identification of the crosslink site. N = any nucleotide; 4SU = 4-Thiouridine; G = Guanosine; Y = Pyrimidine; XL = symbolizes a cross-link position