CFC-seq: identification of full-length capped RNAs unveil enhancer-derived transcription

Yip, C. W.; Parr, C.; Takahashi, H.; Yasuzawa, K.; Valentine, M.; Nishiyori-Sueki, H.; Ugolini, C.; Ranzani, V.; Murata, M.; Kato, M.; Kang, W.; Yip, W. H.; Shibayama, Y.; Sim, A. D.; Chen, Y.; Shu, X.; Moody, J. D.; Umarov, R.; Chang, J.-C.; Pandolfini, L.; Kawashima, T.; Tagami, M.; Nobusada, T.; Kouno, T.; Alfonso Gonzalez, C.; Albanese, R.; Dossena, F.; Haberman, N.; Ozaki, K.; Kasukawa, T.; Lenhard, B.; Frith, M.; Bodega, B.; Nicassio, F.; Calviello, L.; Bienko, M.; Legnini, I.; Hilgers, V.; Gustincich, S.; Goeke, J.; Lecellier, C. H.; Shin, J. W.; Hon, C.-C.; Carninci, P.

doi:10.1101/2024.10.31.620483

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bioRxiv · 10.1101/2024.10.31.620483

CFC-seq: identification of full-length capped RNAs unveil enhancer-derived transcription

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Abstract

Long-read sequencing has transformed transcriptome profiling, yet capturing full-length, non-polyadenylated transcripts like enhancer RNAs (eRNAs) remains challenging. Here, we introduce CFC-seq, combining cap-trapping and in vitro poly(A)-tailing to sequence poly(A) and non-poly(A) RNAs with precise transcription start site. Paired with our assembler, SALA, we identified 39,425 novel transcriptional units, including [~]24,000 eRNAs. Our data reveal a distinct genomic code governing eRNA fate dictated by core promoter architecture. CpG-island enhancers show high chromatin connectivity but yield short, exosome-sensitive RNAs. Conversely, TATA-box enhancers systematically co-opt LTR retrotransposons to inherit structural motifs that produce long, stable, and spliced RNAs. Mechanistically, the pioneer factor NF-Y activates these viral elements to license transcription, balanced by TEAD4 activity across a dual-gear regulatory axis. Finally, non-poly(A) eRNAs terminate via exosome-associated processing at structural-depleted cleavage zones. This comprehensive annotation links enhancer sequence architecture to RNA fate, providing a new transformative framework for decoding the functional human genome. HighlightsO_LIExpanded genomic architecture: CFC-seq unmasks a hidden layer of human transcriptome, identifying 39,425 novel transcriptional units with high-confidence TSS support, including [~]24,000 eRNAs. C_LIO_LITSS-first assembler: We introduce SALA, a specialized long-read assembler that prioritizes authentic 5 Cap-trapped ends to accurately reconstruct the TSS-resolved transcript models. C_LIO_LIGenomic code of eRNA fate: CGI enhancers drive short and exosome-sensitive transcripts associated with repressive H3K27me3 mark and high chromatin connectivity. TATA-box enhancers produce cell-type-specific, long, stable, and frequently spliced eRNAs. C_LIO_LIEvolutionary co-option of retrotransposons: A major fraction of TATA-box eRNAs originate from LTR retrotransposons, providing a direct mechanism for integration of viral elements into the human regulatory landscape. C_LIO_LIA dual-gear pioneering axis: The pioneer factor NF-Y activates unprimed LTR-TATA enhancers to license transcription independent of histone acetylation cascades, operating in parallel with TEAD4-mediated activation. C_LIO_LIStructural determinants of eRNA termination: Non-poly(A) eRNA TES features a secondary structure depletion zone that coordinates pol II termination and calibrates exosome-mediated turnover. C_LI

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Yip, C. W., Parr, C., Takahashi, H., Yasuzawa, K., Valentine, M., Nishiyori-Sueki, H., Ugolini, C., Ranzani, V., Murata, M., Kato, M., Kang, W., Yip, W. H., Shibayama, Y., Sim, A. D., Chen, Y., Shu, X., Moody, J. D., Umarov, R., Chang, J.-C., Pandolfini, L., Kawashima, T., Tagami, M., Nobusada, T., Kouno, T., Alfonso Gonzalez, C., Albanese, R., Dossena, F., Haberman, N., Ozaki, K., Kasukawa, T., Lenhard, B., Frith, M., Bodega, B., Nicassio, F., Calviello, L., Bienko, M., Legnini, I., Hilgers, V., Gustincich, S., Goeke, J., Lecellier, C. H., Shin, J. W., Hon, C.-C., Carninci, P.. 2024-10-31. CFC-seq: identification of full-length capped RNAs unveil enhancer-derived transcription. https://doi.org/10.1101/2024.10.31.620483

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