Introducing HEAP: a new tool for miRNA targets discovery

Our HEAP paper is finally out in Molecular Cell! The result of a close collaboration with Christina Leslie and her group, this work was led by Xiaoyi Li, Yuri Pritykin, and Carla Concepcion. We invite you to read the full paper for all the details, but if you are interested just in the punch line, here is a broad overview.

Xiaoyi, Yuri, and Carla: the brains and the hands behind this work.

MicroRNAs (miRNAs) are evolutionarily conserved small RNAs that mediate post-transcriptional gene repression. They exert the repressive functions through binding to targets, which, in most cases, are the 3’ untranslated regions (3’UTRs) of mRNAs. This interaction is mediated by the miRNA-induced silencing complex (miRISC). The core component of miRISC is the Argonaute (Ago) family protein, which consists of four protein members in mammalian genomes (Ago1-4). Ago2 is the most important member among all Ago proteins (Figure 1). Our group, along with many others, has shown the important roles that miRNAs play under both physiological and pathological conditions 1-3.

Figure 1. The miRISC complex

Over the past two decades a major focus in this field has been to assign phenotypes to individual miRNAs and miRNA families and to define their mechanism(s) of action. Mapping the biological targets of miRNAs has become a key aspect in miRNA research, and a wide range of computational and experimental tools have been developed over the years for miRNA targets determination.

Identification of miRNA targets by crosslinking immunoprecipitation (HITS-CLIP) followed by high-throughput sequencing is the prototype of a class of methods (HITS-CLIP, iCLIP, PAR-CLIP, CLASH, CLEAR-CLIP, eCLIP, etc) 4-9 developed to directly purifying miRNAs and their targets by UV crosslinking and immunoprecipitation of Ago-containing complexes from cells. These methods theoretically provide a transcriptome-wide targeting landscape of all expressed miRNAs in cells.

Although these methods have proven extraordinarily useful in mapping miRNA targets and in learning the rules used by miRNAs to select their targets in various cellular contexts, several intrinsic difficulties have to be overcome before they can be applied to broader cellular contexts, especially, to in vivo contexts. These difficulties include technical complexity and the lack of an in vivo platform which allows flexible targets purification from live tissues/organs.

The motivation of this study was to design a novel strategy to address some of the limitations of CLIP-based methods. To this end we took advantage of the HaloTag system developed by Promega, which offers an easy, antibody-free, approach for protein isolation and labeling 10. The HaloTag is a mutated bacterial haloalkane dehalogenase that catalyzes an irreversible covalent bond between itself and its substrate 11,12. By fusing the HaloTag to Ago2 and using synthetic HaloTag ligands conjugated to beads for complex purification, we developed a simplified miRNA target purification pipeline, built on top of the HITS-CLIP protocol, which can free us from the time-consuming procedures seen in conventional CLIP methods (Figure 2). We named this method Halo-enhanced Ago2 pull-down (HEAP). In the paper we show that the enhanced purification stringency and fewer steps result in miRNA target libraries with great depth, resolution and reproducibility.

Figure 2:outline of the HEAP protocol.

To address the need of in vivo miRNA target purification, we generated a novel genetically engineered mouse model harboring a conditional Halo-Ago2 allele. The HaloTag was knocked in front of Ago2 with a “loxP-STOP-IRES-FLAG-loxP” cassette in between. The Halo-Ago2 fusion is therefore expressed in a Cre-regulated manner, adding another layer of flexibility to this system. We demonstrated the usefulness of this system by identifying targets from several different in vivo contexts, including mouse embryos, adult tissues and primary tumors.

Our group has long-lasting interest in studying the biological functions miR-17~92. To determine the direct targets of members in this miRNA cluster, we generated HEAP libraries from E13.5 embryos lacking miR-17~92. By doing that, we identified a large number of binding sites which contained seed matches for miRNAs in miR-17~92 and whose peak signals decreased when ablating miR-17~92 genetically (Figure 3a). Interestingly we found that a small fraction of Ago2 binding sites mapped to long non-coding RNAs (lncRNAs). One interesting example is Cyrano, a lncRNA containing two miR-92 binding sites (Figure 3b). Differential gene expression analysis in miR-92-deficient mice supports the functionality of these miR-92 binding sites.

Figure 3: HEAP libraries from miR-17~92 mutant embryos.

Lastly, we wanted to look into miRNA regulations under pathological conditions. One interesting setting is primary cancers where massive transcriptomic rewiring has occurred during transformation. To compare miRNA regulations in tumors versus normal cells, we induced primary tumors in the conditional Halo-Ago2 mice and expressed Cre to activate Halo-Ago2 expression. We generated HEAP libraries from two tumor types (glioma and non-small cell lung cancer) and from their corresponding tissues of origin. A direct comparison of miRNA binding sites between tumor versus normal tissues illustrated striking differences between the two. For example, in normal cortex, miRNAs such as miR-124 and miR-128 had large number of targets involved in normal brain physiology, while in tumors, miR-219 became highly “active”, showing the largest number of targets (Figure 4a). We also showed that miRNA abundance was the most likely determinant of miRNA targets seen in each context (Figure 4b). Cross-context comparisons also highlighted the enrichment of miR-17~92 binding sites in both glioma and lung cancers, pointing to a potential general requirement of miR-17~92 for oncogenesis.

Figure 4. HEAP libraries from gliomas and normal cortices.

One aspect we think is important to emphasize is that the Halo-Ago2 mouse strain can be easily employed in conjunction with any of the many already available CLIP variants (PAR-CLIP, eCLIP, iCLIP, CLASH, etc.) to obtain even more granular information on miRNA targets. Finally, the HaloTag provides also an excellent opportunity to identify novel Ago2 interactors and post-translational modifications in vivo, and to image the dynamic of miRISC in living cells.

Video 1, Halo-Ago2 localization in living cells (in collaboration with Ryan Schreiner).

We sincerely hope the scientific community will benefit from this new tool. To facilitate its wide adoption, we are depositing the Halo-Ago2 mouse strain with the Jackson laboratories. Detailed computational pipelines, scripts, and protocols can be found following this link.


Bibliography

1          Ventura, A. et al. Targeted deletion reveals essential and overlapping functions of the miR-17 through 92 family of miRNA clusters. Cell 132, 875-886, doi:10.1016/j.cell.2008.02.019 (2008).

2          Han, Y. C. et al. An allelic series of miR-17 approximately 92-mutant mice uncovers functional specialization and cooperation among members of a microRNA polycistron. Nat Genet 47, 766-775, doi:10.1038/ng.3321 (2015).

3          Bartel, D. P. Metazoan MicroRNAs. Cell 173, 20-51, doi:10.1016/j.cell.2018.03.006 (2018).

4          Chi, S. W., Zang, J. B., Mele, A. & Darnell, R. B. Argonaute HITS-CLIP decodes microRNA-mRNA interaction maps. Nature 460, 479-486, doi:10.1038/nature08170 (2009).

5          Hafner, M. et al. Transcriptome-wide identification of RNA-binding protein and microRNA target sites by PAR-CLIP. Cell 141, 129-141, doi:10.1016/j.cell.2010.03.009 (2010).

6          Helwak, A., Kudla, G., Dudnakova, T. & Tollervey, D. Mapping the human miRNA interactome by CLASH reveals frequent noncanonical binding. Cell 153, 654-665, doi:10.1016/j.cell.2013.03.043 (2013).

7          Konig, J. et al. iCLIP reveals the function of hnRNP particles in splicing at individual nucleotide resolution. Nat Struct Mol Biol 17, 909-915, doi:10.1038/nsmb.1838 (2010).

8          Moore, M. J. et al. miRNA-target chimeras reveal miRNA 3′-end pairing as a major determinant of Argonaute target specificity. Nat Commun 6, 8864, doi:10.1038/ncomms9864 (2015).

9         Van Nostrand, E. L. et al. Robust transcriptome-wide discovery of RNA-binding protein binding sites with enhanced CLIP (eCLIP). Nat Methods 13, 508-514, doi:10.1038/nmeth.3810 (2016).

10        Gu, J. et al. GoldCLIP: Gel-omitted Ligation-dependent CLIP. Genomics, Proteomics & Bioinformatics 16, 136-143, doi:https://doi.org/10.1016/j.gpb.2018.04.003 (2018).

11        Encell, L. P. et al. Development of a dehalogenase-based protein fusion tag capable of rapid, selective and covalent attachment to customizable ligands. Curr Chem Genomics 6, 55-71, doi:10.2174/1875397301206010055 (2012).

12        Los, G. V. et al. HaloTag: a novel protein labeling technology for cell imaging and protein analysis. ACS Chem Biol 3, 373-382, doi:10.1021/cb800025k (2008).

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