The landscape of human p53-regulated long non-coding RNAs reveals critical host gene co-regulation.

The role of long non-coding RNAs (lncRNAs) in p53-mediated tumor suppression has become increasingly appreciated in the past decade. Thus, the identification of p53-regulated lncRNAs can be a promising starting point to select and prioritize lncRNAs for functional analyses. By integrating transcriptome and transcription factor-binding data, we identified 379 lncRNAs that are recurrently differentially regulated by p53. Dissecting the mechanisms by which p53 regulates many of them, we identified sets of lncRNAs regulated either directly by p53 or indirectly through the p53-RFX7 and p53-p21-DREAM/RB:E2F pathways. Importantly, we identified multiple p53-responsive lncRNAs that are co-regulated with their protein-coding host genes, revealing an important mechanism by which p53 may regulate lncRNAs. Further analysis of transcriptome data and clinical data from cancer patients showed that recurrently p53-regulated lncRNAs are associated with patient survival. Together, the integrative analysis of the landscape of p53-regulated lncRNAs provides a powerful resource facilitating the identification of lncRNA function and displays the mechanisms of p53-dependent regulation that could be exploited for developing anticancer approaches.

No Expression Divergence despite Transcriptional Interference between Nested Protein-Coding Genes in Mammals.

Nested protein-coding genes accumulated throughout metazoan evolution, with early analyses of human and Drosophila microarray data indicating that this phenomenon was simply due to the presence of large introns. However, a recent study employing RNA-seq data uncovered evidence of transcriptional interference driving rapid expression divergence between Drosophila nested genes, illustrating that accurate expression estimation of overlapping genes can enhance detection of their relationships. Hence, here I apply an analogous approach to strand-specific RNA-seq data from human and mouse to revisit the role of transcriptional interference in the evolution of mammalian nested genes. A genomic survey reveals that whereas mammalian nested genes indeed accrued over evolutionary time, they are retained at lower frequencies than in Drosophila. Though several properties of mammalian nested genes align with observations in Drosophila and with expectations under transcriptional interference, contrary to both, their expression divergence is not statistically different from that between unnested genes, and also does not increase after nesting. Together, these results support the hypothesis that lower selection efficiencies limit rates of gene expression evolution in mammals, leading to their reliance on immediate eradication of deleterious nested genes to avoid transcriptional interference.

Identification of a unique endoplasmic retention motif in the Xenopus GIRK5 channel and its contribution to oocyte maturation.

G protein-activated inward-rectifying potassium (K+ ) channels (Kir3/GIRK) participate in cell excitability. The GIRK5 channel is present in Xenopus laevis oocytes. In an attempt to investigate the physiological role of GIRK5, we identified a noncanonical di-arginine endoplasmic reticulum (ER) retention motif (KRXY). This retention motif is located at the N-terminal region of GIRK5, coded by two small exons found only in X. laevis and X. tropicalis. These novel exons are expressed through use of an alternative transcription start site. Mutations in the sequence KRXY produced functional channels and induced progesterone-independent oocyte meiotic progression. The chimeric proteins enhanced green fluorescent protein (EGFP)-GIRK5-WT and the EGFP-GIRK5K13AR14A double mutant, were localized to the ER and the plasma membrane of the vegetal pole of the oocyte, respectively. Silencing of GIRK5 or blocking of this channel by external barium prevented progesterone-induced meiotic progression. The endogenous level of GIRK5 protein decreased through oocyte stages in prophase I augmenting by progesterone. In conclusion, we have identified a unique mechanism by which the expression pattern of a K+ channel evolved to control Xenopus oocyte maturation.

Russian Doll Genes and Complex Chromosome Rearrangements in Oxytricha trifallax.

Ciliates have two different types of nuclei per cell, with one acting as a somatic, transcriptionally active nucleus (macronucleus; abbr. MAC) and another serving as a germline nucleus (micronucleus; abbr. MIC). Furthermore, Oxytricha trifallax undergoes extensive genome rearrangements during sexual conjugation and post-zygotic development of daughter cells. These rearrangements are necessary because the precursor MIC loci are often both fragmented and scrambled, with respect to the corresponding MAC loci. Such genome architectures are remarkably tolerant of encrypted MIC loci, because RNA-guided processes during MAC development reorganize the gene fragments in the correct order to resemble the parental MAC sequence. Here, we describe the germline organization of several nested and highly scrambled genes in Oxytricha trifallax These include cases with multiple layers of nesting, plus highly interleaved or tangled precursor loci that appear to deviate from previously described patterns. We present mathematical methods to measure the degree of nesting between precursor MIC loci, and revisit a method for a mathematical description of scrambling. After applying these methods to the chromosome rearrangement maps of O. trifallax we describe cases of nested arrangements with up to five layers of embedded genes, as well as the most scrambled loci in O. trifallax.

Hitting two birds with one stone: The unforeseen consequences of nested gene knockouts in Caenorhabditis elegans.

Nested genes represent an intriguing form of non-random genomic organization in which the boundaries of one gene are fully contained within another, longer host gene. The C. elegans genome contains over 10,000 nested genes, 92% of which are ncRNAs, which occur inside 16% of the protein coding gene complement. Host genes are longer than non-host coding genes, owing to their longer and more numerous introns. Indel alleles are available for nearly all of these host genes that simultaneously alter the nested gene, raising the possibility of nested gene disruption contributing to phenotypes that might be attributed to the host gene. Such dual-knockouts could represent a source of misinterpretation about host gene function. Dual-knockouts might also provide a novel source of synthetic phenotypes that reveal the functional effects of ncRNA genes, whereby the host gene disruption acts as a perturbed genetic background to help unmask ncRNA phenotypes.

Transcriptional Interference Promotes Rapid Expression Divergence of Drosophila Nested Genes.

Nested genes are the most common form of protein-coding overlap in eukaryotic genomes. Previous studies have shown that nested genes accumulate rapidly over evolutionary time, typically via the insertion of short young duplicate genes into long introns. However, the evolutionary relationship between nested genes remains unclear. Here, I compare RNA-seq expression profiles of nested, proximal intra-chromosomal, intermediate intra-chromosomal, distant intra-chromosomal, and inter-chromosomal gene pairs in two Drosophila species. I find that expression profiles of nested genes are more divergent than those of any other class of genes, supporting the hypothesis that concurrent expression of nested genes is deleterious due to transcriptional interference. Further analysis reveals that expression profiles of derived nested genes are more divergent than those of their ancestral un-nested orthologs, which are more divergent than those of un-nested genes with similar genomic features. Thus, gene expression divergence between nested genes is likely caused by selection against nesting of genes with insufficiently divergent expression profiles, as well as by continued expression divergence after nesting. Moreover, expression divergence and sequence evolutionary rates are elevated in young nested genes and reduced in old nested genes, indicating that a burst of rapid evolution occurs after nesting. Together, these findings suggest that similarity between expression profiles of nested genes is deleterious due to transcriptional interference, and that natural selection addresses this problem both by eradicating highly deleterious nestings and by enabling rapid expression divergence of surviving nested genes, thereby quickly limiting or abolishing transcriptional interference.

The evolution and functional significance of nested gene structures in Drosophila melanogaster.

Nearly 10% of the genes in the genome of Drosophila melanogaster are in nested structures, in which one gene is completely nested within the intron of another gene (nested and including gene, respectively). Even though the coding sequences and untranslated regions of these nested/including gene pairs do not overlap, their intimate structures and the possibility of shared regulatory sequences raise questions about the evolutionary forces governing the origination and subsequent functional and evolutionary impacts of these structures. In this study, we show that nested genes experience weaker evolutionary constraint, have faster rates of protein evolution, and are expressed in fewer tissues than other genes, while including genes show the opposite patterns. Surprisingly, despite completely overlapping with each other, nested and including genes are less likely to display correlated gene expression and biological function than the nearby yet nonoverlapping genes. Interestingly, significantly fewer nested genes are transcribed from the same strand as the including gene. We found that same-strand nested genes are more likely to be single-exon genes. In addition, same-strand including genes are less likely to have known lethal or sterile phenotypes than opposite-strand including genes only when the corresponding nested genes have introns. These results support our hypothesis that selection against potential erroneous mRNA splicing when nested and including genes are on the same strand plays an important role in the evolution of nested gene structures.