Protocol to study the inheritance and propagation of non-genetically encoded states using barcode decay lineage tracing.

Here, we present a protocol to perform barcode decay lineage tracing followed by single-cell transcriptome analysis (BdLT-Seq). We describe steps for BdLT-Seq experimental design, building barcoded episome reporters, performing episome transfection, and barcode retrieval. We then describe procedures for sequencing library construction while providing options for sample multiplexing and data analysis. This BdLT-Seq technique enables the assessment of clonal evolution in a directional manner while preserving isogeneity, thus allowing the comparison of non-genetic molecular features between isogenic cell lineages. For complete details on the use and execution of this protocol, please refer to Shlyakhtina et al. (2023).1.

BdLT-Seq as a barcode decay-based method to unravel lineage-linked transcriptome plasticity.

Cell plasticity is a core biological process underlying a myriad of molecular and cellular events taking place throughout organismal development and evolution. It has been postulated that cellular systems thrive to balance the organization of meta-stable states underlying this phenomenon, thereby maintaining a degree of populational homeostasis compatible with an ever-changing environment and, thus, life. Notably, albeit circumstantial evidence has been gathered in favour of the latter conceptual framework, a direct observation of meta-state dynamics and the biological consequences of such a process in generating non-genetic clonal diversity and divergent phenotypic output remains largely unexplored. To fill this void, here we develop a lineage-tracing technology termed Barcode decay Lineage Tracing-Seq. BdLT-Seq is based on episome-encoded molecular identifiers that, supported by the dynamic decay of the tracing information upon cell division, ascribe directionality to a cell lineage tree whilst directly coupling non-genetic molecular features to phenotypes in comparable genomic landscapes. We show that cell transcriptome states are both inherited, and dynamically reshaped following constrained rules encoded within the cell lineage in basal growth conditions, upon oncogene activation and throughout the process of reversible resistance to therapeutic cues thus adjusting phenotypic output leading to intra-clonal non-genetic diversity.

The role of non-genetic information in evolutionary frameworks.

The evolution of organisms has been a subject of paramount debate for hundreds of years and though major advances in the field have been made, the precise mechanisms underlying evolutionary processes remain fragmentary. Strikingly, the majority of the core principles accepted across the many fields of biology only consider genetic information as the major - if not exclusive - biological information carrier and thus consider it as the main evolutionary avatar. However, the real picture appears far more complex than originally anticipated, as compelling data suggest that nongenetic information steps up when highly dynamic evolutionary frameworks are explored. In light of recent evidence, we discuss herein the dynamic nature and complexity of nongenetic information carriers, and their emerging relevance in the evolutionary process. We argue that it is possible to overcome the historical arguments which dismissed these carriers, and instead consider that they are indeed core to life itself as they support a sustainable, continuous source of rapid adaptation in ever-changing environments. Ultimately, we will address the intricacies of genetic and non-genetic networks underlying evolutionary models to build a framework where both core biological information concepts are considered non-negligible and equally fundamental.

Genetic and Non-Genetic Mechanisms Underlying Cancer Evolution.

Cancer development can be defined as a process of cellular and tissular microevolution ultimately leading to malignancy. Strikingly, though this concept has prevailed in the field for more than a century, the precise mechanisms underlying evolutionary processes occurring within tumours remain largely uncharacterized and rather cryptic. Nevertheless, although our current knowledge is fragmentary, data collected to date suggest that most tumours display features compatible with a diverse array of evolutionary paths, suggesting that most of the existing macro-evolutionary models find their avatar in cancer biology. Herein, we discuss an up-to-date view of the fundamental genetic and non-genetic mechanisms underlying tumour evolution with the aim of concurring into an integrated view of the evolutionary forces at play throughout the emergence and progression of the disease and into the acquisition of resistance to diverse therapeutic paradigms. Our ultimate goal is to delve into the intricacies of genetic and non-genetic networks underlying tumour evolution to build a framework where both core concepts are considered non-negligible and equally fundamental.

Asymmetric Inheritance of Cell Fate Determinants: Focus on RNA.

During the last decade, and mainly primed by major developments in high-throughput sequencing technologies, the catalogue of RNA molecules harbouring regulatory functions has increased at a steady pace. Current evidence indicates that hundreds of mammalian RNAs have regulatory roles at several levels, including transcription, translation/post-translation, chromatin structure, and nuclear architecture, thus suggesting that RNA molecules are indeed mighty controllers in the flow of biological information. Therefore, it is logical to suggest that there must exist a series of molecular systems that safeguard the faithful inheritance of RNA content throughout cell division and that those mechanisms must be tightly controlled to ensure the successful segregation of key molecules to the progeny. Interestingly, whilst a handful of integral components of mammalian cells seem to follow a general pattern of asymmetric inheritance throughout division, the fate of RNA molecules largely remains a mystery. Herein, we will discuss current concepts of asymmetric inheritance in a wide range of systems, including prions, proteins, and finally RNA molecules, to assess overall the biological impact of RNA inheritance in cellular plasticity and evolutionary fitness.

TARDIS, a targeted RNA directional sequencing method for rare RNA discovery.

High-throughput transcriptional analysis has unveiled a myriad of novel RNAs. However, technical constraints in RNA sequencing library preparation and platform performance hamper the identification of rare transcripts contained within the RNA repertoire. Herein we present targeted-RNA directional sequencing (TARDIS), a hybridization-based method that allows subsets of RNAs contained within the transcriptome to be interrogated independently of transcript length, function, the presence or absence of poly-A tracts, or the mechanism of biogenesis. TARDIS is a modular protocol that is subdivided into four main phases, including the generation of random DNA traps covering the region of interest, purification of input RNA material, DNA trap-based RNA capture, and finally RNA-sequencing library construction. Importantly, coupling RNA capture to strand-specific RNA sequencing enables robust identification and reconstruction of novel transcripts, the definition of sense and antisense RNA pairs and, by the concomitant analysis of long and natural small RNA pools, it allows the user to infer potential precursor-product relations. TARDIS takes ∼10 d to implement.

Human cells contain natural double-stranded RNAs with potential regulatory functions.

Recent evidence has suggested the existence of sense-antisense transcription in mammals, but the existence of double-stranded RNAs endowed with biological function has remained elusive. Herein we show that hundreds of putative natural double-stranded RNAs (ndsRNAs) are expressed from interspersed genomic locations and respond to cellular cues. We demonstrate that a subset of ndsRNAs localize in the nucleus and, in their double-stranded form, interact with nuclear proteins. Detailed characterization of an ndsRNA (nds-2a) revealed that this molecule displays differential localization throughout the cell cycle and directly interacts with RCC1 and RAN and, through the latter, with the mitotic RANGAP1-SUMO1-RANBP2 complex. Notably, altering nds-2a levels led to postmitotic abnormalities, mitotic catastrophe and cell death, thus supporting a mitosis-related role. Altogether, our study reveals a hitherto-unrecognized class of RNAs that potentially participate in major biological processes in human cells.

MicroRNA-27a regulates basal transcription by targeting the p44 subunit of general transcription factor IIH.

General transcription factor IIH (TFIIH) is a complex RNA polymerase II basal transcription factor comprising 10 different polypeptides that display activities involved in transcription and DNA repair processes. Although biochemical studies have uncovered TFIIH importance, little is known about how the mRNAs that code for TFIIH subunits are regulated. Here it is shown that mRNAs encoding seven of the TFIIH subunits (p34, p44, p52, p62, XPB, CDK7, and p8) are regulated at the posttranscriptional level in a Dicer-dependent manner. Indeed, abolition of the miRNA pathway induces abnormal accumulation, stabilization, and translational activation of these seven mRNAs. Herein, miR-27a was identified as a key regulator of p44 mRNA. Moreover, miR-27a was shown to destabilize the p44 subunit of the TFIIH complex during the G2-M phase, thereby modulating the transcriptional shutdown observed during this transition. This work is unique in providing a demonstration of global transcriptional regulation through the action of a single miRNA.

c-Fos activated phospholipid synthesis is required for neurite elongation in differentiating PC12 cells.

We have previously shown that c-Fos activates phospholipid synthesis through a mechanism independent of its genomic AP-1 activity. Herein, using PC12 cells induced to differentiate by nerve growth factor, the genomic effect of c-Fos in initiating neurite outgrowth is shown as distinct from its nongenomic effect of activating phospholipid synthesis and sustaining neurite elongation. Blocking c-Fos expression inhibited differentiation, phospholipid synthesis activation, and neuritogenesis. In cells primed to grow, blocking c-Fos expression determined neurite retraction. However, transfected cells expressing c-Fos or c-Fos deletion mutants with capacity to activate phospholipid synthesis sustain neurite outgrowth and elongation in the absence of nerve growth factor. Results disclose a dual function of c-Fos: it first releases the genomic program for differentiation and then associates to the endoplasmic reticulum and activates phospholipid synthesis. Because phospholipids are key membrane components, we hypothesize this latter phenomenon as crucial to support membrane genesis demands required for cell growth and neurite elongation.