Juan R. Alvarez-Dominguez

Regulation of mammalian cell differentiation by long non‐coding RNAs

Differentiation of specialized cell types from stem and progenitor cells is tightly regulated at several levels, both during development and during somatic tissue homeostasis. Many long non‐coding RNAs have been recognized as an additional layer of regulation in the specification of cellular identities; these non‐coding species can modulate gene‐expression programmes in various biological contexts through diverse mechanisms at the transcriptional, translational or messenger RNA stability levels. Here, we summarize findings that implicate long non‐coding RNAs in the control of mammalian cell differentiation. We focus on several representative differentiation systems and discuss how specific long non‐coding RNAs contribute to the regulation of mammalian development.

Global discovery of erythroid long noncoding RNAs reveals novel regulators of red cell maturation

Erythropoiesis is regulated at multiple levels to ensure the proper generation of mature red cells under multiple physiological conditions. To probe the contribution of long noncoding RNAs (lncRNAs) to this process, we examined >1 billion RNA-seq reads of polyadenylated and nonpolyadenylated RNA from differentiating mouse fetal liver red blood cells and identified 655 lncRNA genes including not only intergenic, antisense, and intronic but also pseudogene and enhancer loci. More than 100 of these genes are previously unrecognized and highly erythroid specific. By integrating genome-wide surveys of chromatin states, transcription factor occupancy, and tissue expression patterns, we identify multiple lncRNAs that are dynamically expressed during erythropoiesis, show epigenetic regulation, and are targeted by key erythroid transcription factors GATA1, TAL1, or KLF1. We focus on 12 such candidates and find that they are nuclear-localized and exhibit complex developmental expression patterns. Depleting them severely impaired erythrocyte maturation, inhibiting cell size reduction and subsequent enucleation. One of them, alncRNA-EC7, is transcribed from an enhancer and is specifically needed for activation of the neighboring gene encoding BAND 3. Our study provides an annotated catalog of erythroid lncRNAs, readily available through an online resource, and shows that diverse types of lncRNAs participate in the regulatory circuitry underlying erythropoiesis.

Translation of Small Open Reading Frames within Unannotated RNA Transcripts in Saccharomyces cerevisiae

High-throughput gene expression analysis has revealed a plethora of previously undetected transcripts in eukaryotic cells. In this study, we investigate >1,100 unannotated transcripts in yeast predicted to lack protein-coding capacity. We show that a majority of these RNAs are enriched on polyribosomes akin to mRNAs. Ribosome profiling demonstrates that many bind translocating ribosomes within predicted open reading frames 10-96 codons in size. We validate expression of peptides encoded within a subset of these RNAs and provide evidence for conservation among yeast species. Consistent with their translation, many of these transcripts are targeted for degradation by the translation-dependent nonsense-mediated RNA decay (NMD) pathway. We identify lncRNAs that are also sensitive to NMD, indicating that translation of noncoding transcripts also occurs in mammals. These data demonstrate transcripts considered to lack coding potential are bona fide protein coding and expand the proteome of yeast and possibly other eukaryotes.

CASC15-S Is a Tumor Suppressor lncRNA at the 6p22 Neuroblastoma Susceptibility Locus.

Chromosome 6p22 was identified recently as a neuroblastoma susceptibility locus, but its mechanistic contributions to tumorigenesis are as yet undefined. Here we report that the most highly significant single-nucleotide polymorphism (SNP) associations reside within CASC15, a long noncoding RNA that we define as a tumor suppressor at 6p22. Low-level expression of a short CASC15 isoform (CASC15-S) associated highly with advanced neuroblastoma and poor patient survival. In human neuroblastoma cells, attenuating CASC15-S increased cellular growth and migratory capacity. Gene expression analysis revealed downregulation of neuroblastoma-specific markers in cells with attenuated CASC15-S, with concomitant increases in cell adhesion and extracellular matrix transcripts. Altogether, our results point to CASC15-S as a mediator of neural growth and differentiation, which impacts neuroblastoma initiation and progression.

Long noncoding RNAs during normal and malignant hematopoiesis

Long noncoding RNAs (lncRNAs) are increasingly recognized to contribute to cellular development via diverse mechanisms during both health and disease. Here, we highlight recent progress on the study of lncRNAs that function in the development of blood cells. We emphasize lncRNAs that regulate blood cell fates through epigenetic control of gene expression, an emerging theme among functional lncRNAs. Many of these noncoding genes and their targets become dysregulated during malignant hematopoiesis, directly implicating lncRNAs in blood cancers such as leukemia. In a few cases, dysregulation of an lncRNA alone leads to malignant hematopoiesis in a mouse model. Thus, lncRNAs may be not only useful as markers for the diagnosis and prognosis of cancers of the blood, but also as potential targets for novel therapies.

A New System for Comparative Functional Genomics of Saccharomyces Yeasts

Whole-genome sequencing, particularly in fungi, has progressed at a tremendous rate. More difficult, however, is experimental testing of the inferences about gene function that can be drawn from comparative sequence analysis alone. We present a genome-wide functional characterization of a sequenced but experimentally understudied budding yeast, Saccharomyces bayanus var. uvarum (henceforth referred to as S. bayanus), allowing us to map changes over the 20 million years that separate this organism from S. cerevisiae. We first created a suite of genetic tools to facilitate work in S. bayanus. Next, we measured the gene-expression response of S. bayanus to a diverse set of perturbations optimized using a computational approach to cover a diverse array of functionally relevant biological responses. The resulting data set reveals that gene-expression patterns are largely conserved, but significant changes may exist in regulatory networks such as carbohydrate utilization and meiosis. In addition to regulatory changes, our approach identified gene functions that have diverged. The functions of genes in core pathways are highly conserved, but we observed many changes in which genes are involved in osmotic stress, peroxisome biogenesis, and autophagy. A surprising number of genes specific to S. bayanus respond to oxidative stress, suggesting the organism may have evolved under different selection pressures than S. cerevisiae. This work expands the scope of genome-scale evolutionary studies from sequence-based analysis to rapid experimental characterization and could be adopted for functional mapping in any lineage of interest. Furthermore, our detailed characterization of S. bayanus provides a valuable resource for comparative functional genomics studies in yeast.

Self-catalytic DNA Depurination Underlies Human β-Globin Gene Mutations at Codon 6 That Cause Anemias and Thalassemias

Background: A self-catalytic depurination sequence centered at codon 6 in the β-globin gene creates a mutagenic apurinic site. Results: Unique codon 6 haplotypes, many anemia- and thalassemia-causing, far exceed haplotypes at other β-globin codons. Conclusion: Excessive mutagenicity at the only β-globin self-depurination site indicates a mechanism discovered in vitro that functions in vivo. Significance: In vivo functionality of self-depurination sites in genes can spontaneously cause diseases via somatic mutations. The human β-globin gene contains an 18-nucleotide coding strand sequence centered at codon 6 and capable of forming a stem-loop structure that can self-catalyze depurination of the 5′G residue of that codon. The resultant apurinic lesion is subject to error-prone repair, consistent with the occurrence about this codon of mutations responsible for 6 anemias and β-thalassemias and additional substitutions without clinical consequences. The 4-residue loop of this stem-loop-forming sequence shows the highest incidence of mutation across the gene. The loop and first stem base pair-forming residues appeared early in the mammalian clade. The other stem-forming segments evolved more recently among primates, thereby conferring self-depurination capacity at codon 6. These observations indicate a conserved molecular mechanism leading to β-globin variants underlying phenotypic diversity and disease.

Site-Specific Self-Catalyzed DNA Depurination, the Basis of a Spontaneous Mutagenic Mechanism of Wide Evolutionary Significance

This chapter focuses on the nature of site-specific self-catalyzed DNA depurination as a spontaneous mechanism inherent in the chemical structure and dynamics of DNA that has contributed to evolutionary change. It describes the essential molecular features of the mechanism, the short consensus sequence elements that form the catalytic intermediate, the basics of the reactions that lead to the creation of apurinic sites, and the means by which those sites give rise to substitution and short deletion mutations. The consensus sequences are widely distributed in double-stranded genomes across the phyla at high frequency that increases up the phylogenetic tree. In the human genome, they constitute >2 × 106 potential mutagenic sites, non-randomly scattered among very many genes, some containing multiple sites. Examples are presented of genes in which the mutations coincide with their self-depurination consensus sequences, the most striking being those in the β-globin gene that are responsible for six anemias and two β-thalassemias. Those of the olfactory receptor genes and the hypervariable regions of the immunoglobulin genes are shown to have utilized the mechanism to evolve their high degree of diversity and/or to develop their contemporaneous diversity for their present function.

Regulation of Eukaryotic Cell Differentiation by Long Non-coding RNAs

Long noncoding RNAs (lncRNAs) are transcripts longer than 200 nucleotides that do not have functional protein-coding capacity. They can regulate gene expression by affecting the transcription, translation, and stability of mRNA targets through diverse mechanisms. Dozens of eukaryotic lncRNAs have been functionally characterized to date, and they have been associated with important cellular processes such as meiosis, pluripotency, apoptosis, and lineage specification. An emerging theme among known lncRNA functions is therefore the modulation of cell differentiation states, often in response to developmental or environmental cues. This chapter discusses current models of lncRNA function during several well-characterized cell differentiation processes, from yeast to human, highlighting recent evidence that implicate lncRNAs in the regulation of animal development.

Unravelling of the role of long noncoding RNAs in haematopoiesis

The formation of all blood cells requires tight coordination of highly specific yet dynamic gene expression programmes. These programmes are established by integrated networks of transcription factors, chromatin modifiers and regulatory RNAs. Recent advances have revealed that special types of RNAs, long noncoding RNAs (lncRNAs), are critical components in these networks. lncRNAs are activated in cell type‐, developmental stage‐ and context‐specific manners to coordinate gene expression via transcriptional or post‐transcriptional mechanisms. They can function via RNA–DNA, RNA–RNA and RNA–protein interactions and can thus affect the activation of genes or the splicing, stability or translation of their transcripts. As a result, distinct collections of lncRNA regulators help modulate the stepwise development of distinct blood cell types. We highlight recent progress, emerging themes and prospective challenges in unravelling the contributions of lncRNAs to haematopoiesis, as well as their implication in blood diseases such as leukaemia.