Physiological intron retaining transcripts in the cytoplasm abound during human motor neurogenesis Marija Petric ́ Howe, 1,2,6 Hamish Crerar, 1,2,6 Jacob Neeves, 1,2 Jasmine Harley, 1,2 Giulia E. Tyzack, 1,2 Pierre Klein, 1,3 Andres Ramos, 3 Rickie Patani, 1,2,7 and Raphaëlle Luisier 4,5,7 1 The Francis Crick Institute, London NW1 1AT, United Kingdom; 2 Department of Molecular Neuroscience, UCL Institute of Neurology, London WC1N 3AR, United Kingdom; 3 Research Department of Structural and Molecular Biology, University College London, London WC1E 6XA, United Kingdom; 4 Idiap Research Institute, Genomics and Health Informatics, CH-1920 Martigny, Switzerland; 5 SIB Swiss Institute of Bioinformatics, 1015 Lausanne, Switzerland Intron retention (IR) is now recognized as a dominant splicing event during motor neuron (MN) development; however, the role and regulation of intron-retaining transcripts (IRTs) localized to the cytoplasm remain particularly understudied. Here we show that IR is a physiological process that is spatiotemporally regulated during MN lineage restriction and that IRTs in the cytoplasm are detected in as many as 13% (n = 2297) of the genes expressed during this process. We identify a major class of cytoplasmic IRTs that are not associated with reduced expression of their own genes but instead show a high capacity for RNA-binding protein and miRNA occupancy. Finally, we show that ALS-causing VCP mutations lead to a selective increase in cytoplasmic abundance of this particular class of IRTs, which in turn temporally coincides with an increase in the nuclear expression level of predicted miRNA target genes. Altogether, our study identifies a previously unrecognized class of cy- toplasmic intronic sequences with potential regulatory function beyond gene expression. [Supplemental material is available for this article.] Intron retention (IR), a mode of alternative splicing (AS) whereby one or more introns are retained within mature polyadenylated mRNAs, has been greatly understudied in mammalian systems and for a long time was mostly considered as a product of ineffi- cient or erroneous splicing. With advances in detection strategies, IR is now recognized as a more widespread and regulated process than previously thought, and the idea that IR could even function- ally modulate cellular processes has come into focus, with its role(s) in cellular physiology beginning to unfold (Braunschweig et al. 2014; Boutz et al. 2015). Neural cells have a higher proportion of transcripts with re- tained introns compared with other cell types, and an expanding body of evidence suggests a functional role for IR both in neuronal development and homeostasis (Buckley et al. 2011; Yap et al. 2012; Braunschweig et al. 2014; Mauger et al. 2016). Transcripts with IR are often detained in the nucleus as a means of reducing expres- sion levels of transcripts not required for cellular physiology at that particular time (Xu et al. 2008; Boutz et al. 2015; Braun et al. 2017). Some of these transcripts will eventually be degraded by the nuclear exosome, whereas specific signals could stimulate splicing of the retained intron in others, resulting in export of the fully spliced mRNA into the cytoplasm and its subsequent translation (Mauger et al. 2016). Indeed, nuclear detention of in- tron-retaining transcripts (IRTs) provides a powerful mechanism to hold gene expression in a suppressed but poised state that allows rapid protein production if and when an appropriate stimulus is re- ceived (Ninomiya et al. 2011; Kalyna et al. 2012; Yap et al. 2012; Boothby et al. 2013; Mauger et al. 2016). Although the stable cytoplasmic localization of intronic se- quences in neurons has been reported since 2013 (Khaladkar et al. 2013), there has been limited investigation into the possible role of cytoplasmic IRTs. This has presumably been overlooked in part owing to detection limitations but also owing to a notion that these transcripts would likely contain premature translation termination codons (PTCs) and, as such, be degraded by non- sense-mediated mRNA decay (NMD) (Jaillon et al. 2008). Nevertheless, the NMD process is only partially efficient (Lindeboom et al. 2016; Dyle et al. 2020), and conditions exist in which a PTC does not lead to NMD (Lykke-Andersen and Jensen 2015). Although examples of IR coupled with NMD have been found to down-regulate gene expression, such as in granulocyte de- velopment (Wong et al. 2013), these transcripts can also encounter other fates in the cell (Vanichkina et al. 2018). Indeed, one of the few studies focusing on cytoplasmic IR in neurons showed an “ad- dressing” function for intronic RNA sequences, determining the spatial localization of their host transcripts within cellular com- partments such as dendrites (Sharangdhar et al. 2017). Another speculated function of IR has arisen following the finding that re- tained introns can be enriched in miRNA binding sites compared with nonretained introns (Schmitz et al. 2017). This offers an in- triguing route through which miRNA-directed degradation path- ways might regulate the abundance of IRTs; alternatively, the retained introns themselves may serve as miRNA sinks or even en- code novel miRNAs termed “mirtrons” (Schmitz et al. 2017; 6 These authors contributed equally to this work. 7 These authors contributed equally to this work. Corresponding authors: raphaelle.luisier@idiap.ch, rickie.patani@ucl.ac.uk Article published online before print. Article, supplemental material, and publi- cation date are at https://www.genome.org/cgi/doi/10.1101/gr.276898.122. Freely available online through the Genome Research Open Access option. © 2022 Petrić Howe et al. This article, published in Genome Research, is avail- able under a Creative Commons License (Attribution 4.0 International), as de- scribed at http://creativecommons.org/licenses/by/4.0/. Research 1808 Genome Research 32:1808–1825 Published by Cold Spring Harbor Laboratory Press; ISSN 1088-9051/22; www.genome.org www.genome.org Cold Spring Harbor Laboratory Press on September 23, 2023 - Published by genome.cshlp.org Downloaded from