Loss of epigenetic information as a cause of mammalian aging.

All living things experience an increase in entropy, manifested as a loss of genetic and epigenetic information. In yeast, epigenetic information is lost over time due to the relocalization of chromatin-modifying proteins to DNA breaks, causing cells to lose their identity, a hallmark of yeast aging. Using a system called "ICE" (inducible changes to the epigenome), we find that the act of faithful DNA repair advances aging at physiological, cognitive, and molecular levels, including erosion of the epigenetic landscape, cellular exdifferentiation, senescence, and advancement of the DNA methylation clock, which can be reversed by OSK-mediated rejuvenation. These data are consistent with the information theory of aging, which states that a loss of epigenetic information is a reversible cause of aging.

Correction: Rapidly evolving protointrons in Saccharomyces genomes revealed by a hungry spliceosome.

[This corrects the article DOI: 10.1371/journal.pgen.1008249.].

Rapidly evolving protointrons in Saccharomyces genomes revealed by a hungry spliceosome.

Introns are a prevalent feature of eukaryotic genomes, yet their origins and contributions to genome function and evolution remain mysterious. In budding yeast, repression of the highly transcribed intron-containing ribosomal protein genes (RPGs) globally increases splicing of non-RPG transcripts through reduced competition for the spliceosome. We show that under these "hungry spliceosome" conditions, splicing occurs at more than 150 previously unannotated locations we call protointrons that do not overlap known introns. Protointrons use a less constrained set of splice sites and branchpoints than standard introns, including in one case AT-AC in place of GT-AG. Protointrons are not conserved in all closely related species, suggesting that most are not under positive selection and are fated to disappear. Some are found in non-coding RNAs (e. g. CUTs and SUTs), where they may contribute to the creation of new genes. Others are found across boundaries between noncoding and coding sequences, or within coding sequences, where they offer pathways to the creation of new protein variants, or new regulatory controls for existing genes. We define protointrons as (1) nonconserved intron-like sequences that are (2) infrequently spliced, and importantly (3) are not currently understood to contribute to gene expression or regulation in the way that standard introns function. A very few protointrons in S. cerevisiae challenge this classification by their increased splicing frequency and potential function, consistent with the proposed evolutionary process of "intronization", whereby new standard introns are created. This snapshot of intron evolution highlights the important role of the spliceosome in the expansion of transcribed genomic sequence space, providing a pathway for the rare events that may lead to the birth of new eukaryotic genes and the refinement of existing gene function.

Competition between pre-mRNAs for the splicing machinery drives global regulation of splicing.

During meiosis in yeast, global splicing efficiency increases and then decreases. Here we provide evidence that splicing improves due to reduced competition for the splicing machinery. The timing of this regulation corresponds to repression and reactivation of ribosomal protein genes (RPGs) during meiosis. In vegetative cells, RPG repression by rapamycin treatment also increases splicing efficiency. Downregulation of the RPG-dedicated transcription factor gene IFH1 genetically suppresses two spliceosome mutations, prp11-1 and prp4-1, and globally restores splicing efficiency in prp4-1 cells. We conclude that the splicing apparatus is limiting and that pre-messenger RNAs compete. Splicing efficiency of a pre-mRNA therefore depends not just on its own concentration and affinity for limiting splicing factor(s), but also on those of competing pre-mRNAs. Competition between RNAs for limiting processing factors appears to be a general condition in eukaryotes for a variety of posttranscriptional control mechanisms including microRNA (miRNA) repression, polyadenylation, and splicing.

Integration of a splicing regulatory network within the meiotic gene expression program of Saccharomyces cerevisiae.

Splicing regulatory networks are essential components of eukaryotic gene expression programs, yet little is known about how they are integrated with transcriptional regulatory networks into coherent gene expression programs. Here we define the MER1 splicing regulatory network and examine its role in the gene expression program during meiosis in budding yeast. Mer1p splicing factor promotes splicing of just four pre-mRNAs. All four Mer1p-responsive genes also require Nam8p for splicing activation by Mer1p; however, other genes require Nam8p but not Mer1p, exposing an overlapping meiotic splicing network controlled by Nam8p. MER1 mRNA and three of the four Mer1p substrate pre-mRNAs are induced by the transcriptional regulator Ume6p. This unusual arrangement delays expression of Mer1p-responsive genes relative to other genes under Ume6p control. Products of Mer1p-responsive genes are required for initiating and completing recombination and for activation of Ndt80p, the activator of the transcriptional network required for subsequent steps in the program. Thus, the MER1 splicing regulatory network mediates the dependent relationship between the UME6 and NDT80 transcriptional regulatory networks in the meiotic gene expression program. This study reveals how splicing regulatory networks can be interlaced with transcriptional regulatory networks in eukaryotic gene expression programs.

miR-122 removal in the liver activates imprinted microRNAs and enables more effective microRNA-mediated gene repression.

miR-122 is a highly expressed liver microRNA that is activated perinatally and aids in regulating cholesterol metabolism and promoting terminal differentiation of hepatocytes. Disrupting expression of miR-122 can re-activate embryo-expressed adult-silenced genes, ultimately leading to the development of hepatocellular carcinoma (HCC). Here we interrogate the liver transcriptome at various time points after genomic excision of miR-122 to determine the cellular consequences leading to oncogenesis. Loss of miR-122 leads to specific and progressive increases in expression of imprinted clusters of microRNAs and mRNA transcripts at the Igf2 and Dlk1-Dio3 loci that could be curbed by re-introduction of exogenous miR-122. mRNA targets of other abundant hepatic microRNAs are functionally repressed leading to widespread hepatic transcriptional de-regulation. Together, this reveals a transcriptomic framework for the hepatic response to loss of miR-122 and the outcome on other microRNAs and their cognate gene targets.

RNA interference-induced hepatotoxicity results from loss of the first synthesized isoform of microRNA-122 in mice.

Small RNAs can be engineered to target and eliminate expression of disease-causing genes or infectious viruses, resulting in the preclinical and clinical development of RNA interference (RNAi) therapeutics using these small RNAs. To ensure the success of RNAi therapeutics, small hairpin RNAs (shRNAs) must co-opt sufficient quantities of the endogenous microRNA machinery to elicit efficient gene knockdown without impeding normal cellular function. We previously observed liver toxicity-including hepatocyte turnover, loss of gene repression and lethality-in mice receiving high doses of a recombinant adeno-associated virus (rAAV) vector expressing shRNAs (rAAV-shRNAs); however the mechanism by which toxicity ensues has not been elucidated. Using rAAV-shRNAs we have now determined that hepatotoxicity arises when exogenous shRNAs exceed 12% of the total amount of liver microRNAs. After this threshold was surpassed, shRNAs specifically reduced the initially synthesized 22-nucleotide isoform of microRNA (miR)-122-5p without substantially affecting other microRNAs, resulting in functional de-repression of miR-122 target mRNAs. Delivery of a rAAV-shRNA vector expressing mature miR-122-5p could circumvent toxicity, despite the exogenous shRNA accounting for 70% of microRNAs. Toxicity was also not observed in Mir122-knockout mice regardless of the level or sequence of the shRNA. Our study establishes limits to the microRNA machinery that is available for therapeutic siRNAs and suggests new paradigms for the role of miR-122 in liver homeostasis in mice.