One-shot analysis of translated mammalian lncRNAs with AHARIBO
Abstract
A vast portion of the mammalian genome is transcribed as long non-coding RNAs (lncRNAs) acting in the cytoplasm with largely unknown functions. Surprisingly, lncRNAs have been shown to interact with ribosomes, encode peptides, or act as ribosome sponges. These functions still remain mostly undetected and understudied owing to the lack of efficient tools for genome-wide simultaneous identification of ribosome-associated and peptide-producing lncRNAs. Here we present AHARIBO, a method for the detection of lncRNAs either untranslated, but associated with ribosomes, or encoding small peptides. Using AHARIBO in mouse embryonic stem cells during neuronal differentiation, we isolated ribosome-protected RNA fragments, translated RNAs and corresponding de novo synthesized peptides. Besides identifying mRNAs under active translation and associated ribosomes, we found and distinguished lncRNAs acting as ribosome sponges or encoding micropeptides, laying the ground for a better functional understanding of hundreds lncRNAs.
Data availability
All data generated or analysed during this study are included in the manuscript and supporting files. Source data files have been provided. All sequencing data are deposited in public archives and made available upon publication.
-
The Translational Landscape of the Human HeartPublicly available and interactive web application for exploring the results of this paper, http://shiny.mdc-berlin.de/cardiac-translatome/.
Article and author information
Author details
Funding
Autonumus Province of Trento adn Banca Intesa (LP6/99)
- Luca Minati
- Claudia Firrito
- Alessia Del Piano
- Alberto Peretti
- Paola Bernabo
- Massimiliano Clamer
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Reviewing Editor
- Howard Y Chang, Stanford University, United States
Publication history
- Received: May 25, 2020
- Accepted: February 16, 2021
- Accepted Manuscript published: February 17, 2021 (version 1)
- Version of Record published: March 4, 2021 (version 2)
Copyright
© 2021, Minati et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
Metrics
-
- 1,985
- Page views
-
- 358
- Downloads
-
- 7
- Citations
Article citation count generated by polling the highest count across the following sources: Crossref, PubMed Central, Scopus.
Download links
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
Further reading
-
- Cell Biology
- Cancer Biology
Recent studies reveal that lateral mitochondrial transfer, the movement of mitochondria from one cell to another, can affect cellular and tissue homeostasis. Most of what we know about mitochondrial transfer stems from bulk cell studies and have led to the paradigm that functional transferred mitochondria restore bioenergetics and revitalize cellular functions to recipient cells with damaged or non-functional mitochondrial networks. However, we show that mitochondrial transfer also occurs between cells with functioning endogenous mitochondrial networks, but the mechanisms underlying how transferred mitochondria can promote such sustained behavioral reprogramming remain unclear. We report that unexpectedly, transferred macrophage mitochondria are dysfunctional and accumulate reactive oxygen species in recipient cancer cells. We further discovered that reactive oxygen species accumulation activates ERK signaling, promoting cancer cell proliferation. Pro-tumorigenic macrophages exhibit fragmented mitochondrial networks, leading to higher rates of mitochondrial transfer to cancer cells. Finally, we observe that macrophage mitochondrial transfer promotes tumor cell proliferation in vivo. Collectively these results indicate that transferred macrophage mitochondria activate downstream signaling pathways in a ROS-dependent manner in cancer cells, and provide a model of how sustained behavioral reprogramming can be mediated by a relatively small amount of transferred mitochondria in vitro and in vivo.
-
- Cell Biology
- Developmental Biology
Environmental cues, such as physical forces and heterotypic cell interactions play a critical role in cell function, yet their collective contributions to transcriptional changes are unclear. Focusing on human endothelial cells, we performed broad individual sample analysis to identify transcriptional drifts associated with environmental changes that were independent of genetic background. Global gene expression profiling by RNAseq and protein expression by LC-MS directed proteomics distinguished endothelial cells in vivo from genetically matched culture (in vitro) samples. Over 43% of the transcriptome was significantly changed by the in vitro environment. Subjecting cultured cells to long-term shear stress significantly rescued the expression of approximately 17% of genes. Inclusion of heterotypic interactions by co-culture of endothelial cells with smooth muscle cells normalized approximately 9% of the original in vivo signature. We also identified novel flow dependent genes, as well as genes that necessitate heterotypic cell interactions to mimic the in vivo transcriptome. Our findings highlight specific genes and pathways that rely on contextual information for adequate expression from those that are agnostic of such environmental cues.