Michiel de Hoon1, Alessandro Bonetti1,12,13, Charles Plessy1,12, Yoshinari Ando1, Chung-Chau Hon1, Yuri Ishizu2, Masayoshi Itoh1,3, Sachi Kato1, Dongyan Lin1,4,5, Sho Maekawa6, Mitsuyoshi Murata1, Hiromi Nishiyori1, Jay W. Shin1,7, Jens Stolte2, Ana Maria Suzuki1, Michihira Tagami1, Hazuki Takahashi1, Supat Thongjuea2, Alistair R.R. Forrest1,8, Yoshihide Hayashizaki3,6, Juha Kere9,10 and Piero Carninci1,11 1RIKEN Center for Integrative Medical Sciences, Yokohama, Kanagawa 230-0045, Japan; 2RIKEN Center for Life Science Technologies, Division of Genomic Technologies, Yokohama, Kanagawa 230-0045, Japan; 3RIKEN Preventive Medicine and Diagnosis Innovation Program, Wako, Saitama 351-0198, Japan; 4Integrated Program in Neuroscience, McGill University, Montreal, Quebec H3A 1A1, Canada; 5Mila, Montreal, Quebec H2S 3H1, Canada; 6RIKEN Omics Science Center (OSC), Yokohama, Kanagawa 230-0045, Japan; 7Genome Institute of Singapore, Agency for Science, Technology and Research (A*STAR), Singapore, 138632, Singapore; 8Harry Perkins Institute of Medical Research, QEII Medical Centre and Centre for Medical Research, The University of Western Australia, Nedlands, Perth, Western Australia 6009, Australia; 9Department of Biosciences and Nutrition, Karolinska Institutet, Huddinge 14157, Sweden; 10Stem Cells and Metabolism Research Program, University of Helsinki and Folkhälsan Research Center, Helsinki 00290, Finland; 11Human Technopole, Milan 20157, Italy

↵12 These authors contributed equally to this work.

↵13 Present address: AstraZeneca Discovery Sciences, Translational Genomics, 43183 Mölndal, Sweden

Corresponding author: michiel.dehoonriken.jp Abstract

In eukaryotes, capped RNAs include long transcripts such as messenger RNAs and long noncoding RNAs, as well as shorter transcripts such as spliceosomal RNAs, small nucleolar RNAs, and enhancer RNAs. Long capped transcripts can be profiled using cap analysis gene expression (CAGE) sequencing and other methods. Here, we describe a sequencing library preparation protocol for short capped RNAs, apply it to a differentiation time course of the human cell line THP-1, and systematically compare the landscape of short capped RNAs to that of long capped RNAs. Transcription initiation peaks associated with genes in the sense direction have a strong preference to produce either long or short capped RNAs, with one out of six peaks detected in the short capped RNA libraries only. Gene-associated short capped RNAs have highly specific 3′ ends, typically overlapping splice sites. Enhancers also preferentially generate either short or long capped RNAs, with 10% of enhancers observed in the short capped RNA libraries only. Enhancers producing either short or long capped RNAs show enrichment for GWAS-associated disease SNPs. We conclude that deep sequencing of short capped RNAs reveals new families of noncoding RNAs and elucidates the diversity of transcripts generated at known and novel promoters and enhancers.

Footnotes

[Supplemental material is available for this article.]

Article published online before print. Article, supplemental material, and publication date are at https://www.genome.org/cgi/doi/10.1101/gr.276647.122.

Freely available online through the Genome Research Open Access option.

Received January 28, 2022. Accepted August 9, 2022.