The approach is based on a peptide (T6B) previously generated by Gunter Meister and Tom Tuschl that binds to Argonaute proteins and prevents the assembly of a functional miRISC complex, thus effectively blocking miRNA-mediated gene repression without impairing miRNA biogenesis.
To explore the consequences of miRNA-inhibition in vivo, we have generated a novel genetically engineered mouse strain that expresses the T6B peptide fused to YFP in a doxycycline-inducible manner. By crossing it with an appropriate rtTA expressing strain it is possible to block miRNA function in a tissue-restricted and temporally controlled manner.
The effect is fully reversible, and miRNA biogenesis (or endogenous siRNA function) are not affected. The entity of de-repression of predicted miRNA targets is roughly proportional to the corresponding miRNA family abundance.
Each “bubble” in the plot above is a set of predicted targets of a miRNA family. On the x axis we plotted the average log2 fold change compared to the background(reported as z-score) and on the y-axis is the miRNA family abundance. Using this novel mouse strain we show that although miRNA-mediated repression is required during embryonic development, in many adult tissues it seems largely dispensable under homeostatic conditions, and becomes essential only in after acute damage and during regeneration.
A notable exception is represented by the skeletal muscle and the heart, where even under physiologic conditions T6B induction results in tissue degeneration and death.
We have a ton of follow-up experiments planned and we hope our colleagues interested in the biology of miRNAs will find it useful. As always, the mouse strain will be freely available through @jacksonlab as soon as we have completed the donation process.
Dr. Gaspare La Rocca deserves a special mention as he was the main driver of this project. He came up with the idea, performed—together with Bryan King—most of the key experiments, and persisted through many ups and downs during the past few years.
A warm welcome to Davide Pradella, our new postdoc!
Davide is from Gravedona, a small village on the north shores of Lake Como, in Italy. He obtained his Ph.D. in Genetics, Molecular, and Cellular Biology from the University of Pavia, working on RNA metabolism at the Institute of Molecular Genetics (IGM).
Davide research interests will focus on ecDNAs, cancer, and gene fusions. Stay tuned!
A big congratulations to our own Xiaoyi Li who today brilliantly earned her PhD with a wonderful presentation to cap five years of hard work and a remarkable first author paper in Molecular Cell. Xiaoyi will soon move to the west coast to start her postdoctoral training.
Running a lab can be frustrating at times, but the privilege of witnessing a young student like Xiaoyi flourish and blossom into an accomplished scientist is, at the end of the day, what makes our job the best in the world.
Ad majora Xiaoyi, the future is yours! And thanks again for choosing us five years ago…
We have an NIH-funded postdoctoral position available immediately to study BRAF-driven pediatric brain cancers. You will use ex vivo and in vivo somatic chromosomal engineering to model and characterize pediatric brain tumors driven by the BRAF oncogene, with a particular focus on tumors driven by BRAF gene fusions.
The goals include defining the molecular mechanisms driving these tumors and responsible for their unique biological and clinical features, and developing and testing novel therapeutic strategies.
Contact us for additional details and check out our work on CRISPR-based chromosomal engineering.
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.
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.
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.
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.
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.
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.
1 Ventura, A. et al. Targeted deletion reveals essential and overlapping functions of the miR-17 through 92 family of miRNA clusters. Cell132, 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 Genet47, 766-775, doi:10.1038/ng.3321 (2015).
3 Bartel, D. P. Metazoan MicroRNAs. Cell173, 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. Nature460, 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. Cell141, 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. Cell153, 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 Biol17, 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 Commun6, 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 Methods13, 508-514, doi:10.1038/nmeth.3810 (2016).
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 Genomics6, 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 Biol3, 373-382, doi:10.1021/cb800025k (2008).
We are happy to announce that our former postdoc Joana Vidigal is starting her own lab at NCI. Given her successes as a grad student and as a postdoc it is easy to predict she will be an outstanding group leader. If you are interested in RNA biology and genome editing, her lab is the place to be. For more information, visit Joana’s website.
Ping Mu, our very first graduate student, after completing a very successful postdoc with Charles Sawyers, has now started his own lab at UTSW where he will continue his ground breaking work on prostate cancer. If you are looking for a postdoctoral position I cannot think of a better environment and a better lab! To learn more visit his lab website or contact Ping via email
Great news today: Alexander Perez was named to the Forbes 30 under 30 list!
This is a prestigious and well-deserved recognition for the important work on CRISPR-Cas9 genome editing he did as a joint MD/PhD student between Christina Leslie’s lab and ours. Way to go Alex!!!
We are proud to announce that our latest manuscript: “Somatic chromosomal engineering identifies BCAN-NTRK1 as a potent glioma driver and therapeutic target”, has been just published in Nature Communications. This work was the result of a truly collaborative effort between our lab and the lab ot our neighbors at MSKCC: Robert Benezra and Maurizio Scaltriti. A follow-up to Danilo Maddalo’s work, this project used CRISPR-based somatic genome editing to model a set of rare genomic rearrangements responsible for brain cancer-associated gene fusions. Peter J. Cook, is the lead author of this paper and is a joint postdoctoral fellow between the Ventura and the Benezra lab, and the project was funded by the Pershing Square Sohn Cancer Foundation and by the Brain Tumor Center at MSKCC.
Peter’s initial goal was to use CRISPR-Cas9 to model a set of uncharacterized, potentially oncogenic chromosomal rearrangements in the brain. Four gene fusions identified from human glioma patient RNA-Seq data were selected and the chromosomal inversions, deletions, and duplications underlying these fusions were modeled in mouse adult neural stem cell primary cultures and directly in the brain using adenovirus-mediated in vivo genome editing. He found that a chromosomal deletion resulting in the fusion between the secreted proteoglycan Bcan and the receptor tyrosine kinase Ntrk1 was capable of transforming p53-null neuronal stem cells cells, resulting in high grade gliomas by either in-vitro orthotopic stem cell implantation or by direct in-vivo viral induction. Interestingly, we also found that these tumors were sensitive to a small molecule kinase inhibitor specific to Ntrk1.
We are excited about this work not only because it resulted in a new a clinically relevant genetically-engineered mouse model for human glioma, but also because it provides an experimental pipeline that can be readily adapted to interrogate a wide range of brain cancer associated mutations of unknown functional significance.