Evidence for the biogenesis of more than 1,000 novel human microRNAs

Background: MicroRNAs (miRNAs) are established regulators of development, cell identity and disease. Although nearly two thousand human miRNA genes are known and new ones are continuously discovered, no attempt has been made to gauge the total miRNA content of the human genome. Results: Employing an innovative computational method on massively pooled small RNA sequencing data, we report 2,469 novel human miRNA candidates of which 1,098 are validated by in-house and published experiments. Almost 300 candidates are robustly expressed in a neuronal cell system and are regulated during differentiation or when biogenesis factors Dicer, Drosha, DGCR8 or Ago2 are silenced. To improve expression profiling, we devised a quantitative miRNA capture system. In a kidney cell system, 400 candidates interact with DGCR8 at transcript positions that suggest miRNA hairpin recognition, and 1,000 of the new miRNA candidates interact with Ago1 or Ago2, indicating that they are directly bound by miRNA effector proteins. From kidney cell CLASH experiments, in which miRNA-target pairs are ligated and sequenced, we observe hundreds of interactions between novel miRNAs and mRNA targets. The novel miRNA candidates are specifically but lowly expressed, raising the possibility that not all may be functional. Interestingly, the majority are evolutionarily young and overrepresented in the human brain. Conclusions: In summary, we present evidence that the complement of human miRNA genes is substantially larger than anticipated, and that more are likely to be discovered in the future as more tissues and experimental conditions are sequenced to greater depth.This project was funded by the Spanish Plan Nacional SAF2008-00357 (NOVADIS); the Generalitat de Catalunya AGAUR 2009 SGR-1502; the Instituto de Salud Carlos III (FIS/FEDER PI11/00733); National Institutes of Health (R00HG004515 to KCC) and the European Commission 7th Framework Program, Projects N. 03790 (SIROCCO), N. 282510 (BLUEPRINT), N. 261123 (GEUVADIS) and N. 262055 (ESGI). MRF is supported by EMBO Long-Term fellowship ALTF 225–2011; EL is supported by the ICGC CLL-Genome project funded by the Spanish Ministry of Economy and Competiveness through the Instituto de Salud Carlos III. AJSH is a Marie Curie postdoctoral fellow supported by the European Commission 7th Framework Program under grant agreement N. 330133. MB-C is a Sara Borrell postdoctoral fellow supported by the Spanish Ministry of Economy and Competiveness. GK was supported by the Wellcome Trust Grant 097383 and by the MRC. EM–H is a PhD student from LaCaix