Amino acid transporter SLC7A5 regulates cell proliferation and secretary cell differentiation and distribution in the mouse intestine.

The intestine is critical for not only processing nutrients but also protecting the organism from the environment. These functions are mainly carried out by the epithelium, which is constantly being self-renewed. Many genes and pathways can influence intestinal epithelial cell proliferation. Among them is mTORC1, whose activation increases cell proliferation. Here, we report the first intestinal epithelial cell (IEC)-specific knockout (ΔIEC) of an amino acid transporter capable of activating mTORC1. We show that the transporter, SLC7A5, is highly expressed in mouse intestinal crypt and Slc7a5ΔIEC reduces mTORC1 signaling. Surprisingly, adult Slc7a5ΔIEC intestinal crypts have increased cell proliferation but reduced mature Paneth cells. Goblet cells, the other major secretory cell type in the small intestine, are increased in the crypts but reduced in the villi. Analyses with scRNA-seq and electron microscopy have revealed dedifferentiation of Paneth cells in Slc7a5ΔIEC mice, leading to markedly reduced secretory granules with little effect on Paneth cell number. Thus, SLC7A5 likely regulates secretory cell differentiation to affect stem cell niche and indirectly regulate cell proliferation.

Yeast eIF2A has a minimal role in translation initiation and uORF-mediated translational control in vivo.

Initiating translation of most eukaryotic mRNAs depends on recruitment of methionyl initiator tRNA (Met-tRNAi) in a ternary complex (TC) with GTP-bound eukaryotic initiation factor 2 (eIF2) to the small (40S) ribosomal subunit, forming a 43S preinitiation complex (PIC) that attaches to the mRNA and scans the 5'-untranslated region (5' UTR) for an AUG start codon. Previous studies have implicated mammalian eIF2A in GTP-independent binding of Met-tRNAi to the 40S subunit and its recruitment to specialized mRNAs that do not require scanning, and in initiation at non-AUG start codons, when eIF2 function is attenuated by phosphorylation of its α-subunit during stress. The role of eIF2A in translation in vivo is poorly understood however, and it was unknown whether the conserved ortholog in budding yeast can functionally substitute for eIF2. We performed ribosome profiling of a yeast deletion mutant lacking eIF2A and isogenic wild-type (WT) cells in the presence or absence of eIF2α phosphorylation induced by starvation for amino acids isoleucine and valine. Whereas starvation of WT confers changes in translational efficiencies (TEs) of hundreds of mRNAs, the eIF2AΔ mutation conferred no significant TE reductions for any mRNAs in non-starved cells, and it reduced the TEs of only a small number of transcripts in starved cells containing phosphorylated eIF2α. We found no evidence that eliminating eIF2A altered the translation of mRNAs containing putative internal ribosome entry site (IRES) elements, or harboring uORFs initiated by AUG or near-cognate start codons, in non-starved or starved cells. Thus, very few mRNAs (possibly only one) appear to employ eIF2A for Met-tRNAi recruitment in yeast cells, even when eIF2 function is attenuated by stress.

Yeast eIF2A has a minimal role in translation initiation and uORF-mediated translational control in vivo.

Initiating translation of most eukaryotic mRNAs depends on recruitment of methionyl initiator tRNA (Met-tRNAi) in a ternary complex (TC) with GTP-bound eukaryotic initiation factor 2 (eIF2) to the small (40S) ribosomal subunit, forming a 43S preinitiation complex (PIC) that attaches to the mRNA and scans the 5'-untranslated region (5' UTR) for an AUG start codon. Previous studies have implicated mammalian eIF2A in GTP-independent binding of Met-tRNAi to the 40S subunit and its recruitment to specialized mRNAs that do not require scanning, and in initiation at non-AUG start codons, when eIF2 function is attenuated by phosphorylation of its α-subunit during stress. The role of eIF2A in translation in vivo is poorly understood however, and it was unknown whether the conserved ortholog in budding yeast can functionally substitute for eIF2. We performed ribosome profiling of a yeast deletion mutant lacking eIF2A and isogenic wild-type (WT) cells in the presence or absence of eIF2α phosphorylation induced by starvation for amino acids isoleucine and valine. Whereas starvation of WT confers changes in translational efficiencies (TEs) of hundreds of mRNAs, the eIF2AΔ mutation conferred no significant TE reductions for any mRNAs in non-starved cells, and it reduced the TEs of only a small number of transcripts in starved cells containing phosphorylated eIF2α. We found no evidence that eliminating eIF2A altered the translation of mRNAs containing putative IRES elements, or harboring uORFs initiated by AUG or near-cognate start codons, in non-starved or starved cells. Thus, very few mRNAs (possibly only one) appear to employ eIF2A for Met-tRNAi recruitment in yeast cells, even when eIF2 function is attenuated by stress.

σ28-dependent small RNA regulation of flagella biosynthesis.

Flagella are important for bacterial motility as well as for pathogenesis. Synthesis of these structures is energy intensive and, while extensive transcriptional regulation has been described, little is known about the posttranscriptional regulation. Small RNAs (sRNAs) are widespread posttranscriptional regulators, most base pairing with mRNAs to affect their stability and/or translation. Here, we describe four UTR-derived sRNAs (UhpU, MotR, FliX and FlgO) whose expression is controlled by the flagella sigma factor σ28 (fliA) in Escherichia coli. Interestingly, the four sRNAs have varied effects on flagellin protein levels, flagella number and cell motility. UhpU, corresponding to the 3´ UTR of a metabolic gene, likely has hundreds of targets including a transcriptional regulator at the top flagella regulatory cascade connecting metabolism and flagella synthesis. Unlike most sRNAs, MotR and FliX base pair within the coding sequences of target mRNAs and act on ribosomal protein mRNAs connecting ribosome production and flagella synthesis. The study shows how sRNA-mediated regulation can overlay a complex network enabling nuanced control of flagella synthesis.

Liver development during Xenopus tropicalis metamorphosis is controlled by T3-activation of WNT signaling.

Thyroid hormone (T3) regulates vertebrate organ development, growth, and metabolism through the T3 receptor (TR). Due to maternal influence in mammals, it has been difficult to study if and how T3 regulates liver development. Liver remodeling during anuran metamorphosis resembles liver maturation in mammals and is controlled by T3. We generated Xenopus tropicalis animals with both TRα and TRß genes knocked out and found that TR double knockout liver had developmental defects such as reduced cell proliferation and failure to undergo hepatocyte hypertrophy or activate urea cycle gene expression. RNA-seq analysis showed that T3 activated canonical Wnt pathway in the liver. Particularly, Wnt11 was activated in both fibroblasts and hepatic cells, and in turn, likely promoted the proliferation and maturation of hepatocytes. Our study offers new insights into not only how T3 regulates liver development but also on potential means to improve liver regeneration.

Differential requirements for Gcn5 and NuA4 HAT activities in the starvation-induced versus basal transcriptomes.

The histone acetyltransferase (HAT) subunit of coactivator complex SAGA, Gcn5, stimulates eviction of promoter nucleosomes at certain highly expressed yeast genes, including those activated by transcription factor Gcn4 in amino acid-deprived cells; however, the importance of other HAT complexes in this process was poorly understood. Analyzing mutations that disrupt the integrity or activity of HAT complexes NuA4 or NuA3, or HAT Rtt109, revealed that only NuA4 acts on par with Gcn5, and functions additively, in evicting and repositioning promoter nucleosomes and stimulating transcription of starvation-induced genes. NuA4 is generally more important than Gcn5, however, in promoter nucleosome eviction, TBP recruitment, and transcription at most other genes expressed constitutively. NuA4 also predominates over Gcn5 in stimulating TBP recruitment and transcription of genes categorized as principally dependent on the cofactor TFIID versus SAGA, except for the most highly expressed subset including ribosomal protein genes, where Gcn5 contributes strongly to PIC assembly and transcription. Both SAGA and NuA4 are recruited to promoter regions of starvation-induced genes in a manner that might be feedback controlled by their HAT activities. Our findings reveal an intricate interplay between these two HATs in nucleosome eviction, PIC assembly, and transcription that differs between the starvation-induced and basal transcriptomes.

Amino acid transporter SLC7A5 regulates Paneth cell function to affect the intestinal inflammatory response.

The intestine is critical for not only processing and resorbing nutrients but also protecting the organism from the environment. These functions are mainly carried out by the epithelium, which is constantly being self-renewed. Many genes and pathways can influence intestinal epithelial cell proliferation. Among them is mTORC1, whose activation increases cell proliferation. Here, we report the first intestinal epithelial cell-specific knockout ( ΔIEC ) of an amino acid transporter capable of activating mTORC1. We show that the transporter, SLC7A5, is highly expressed in mouse intestinal crypt and Slc7a5 ΔIEC reduces mTORC1 signaling. Surprisingly, Slc7a5 ΔIEC mice have increased cell proliferation but reduced secretory cells, particularly mature Paneth cells. scRNA-seq and electron microscopic analyses revealed dedifferentiation of Paneth cells in Slc7a5 ΔIEC mice, leading to markedly reduced secretory granules with little effect on Paneth cell number. We further show that Slc7a5 ΔIEC mice are prone to experimental colitis. Thus, SLC7A5 regulates secretory cell differentiation to affect stem cell niche and/or inflammatory response to regulate cell proliferation.

Comparative Analysis of Transcriptome Profiles Reveals Distinct and Organ-Dependent Genomic and Nongenomic Actions of Thyroid Hormone in Xenopus tropicalis Tadpoles.

Background: Thyroid hormone (triiodothyronine [T3]) is essential for development and organ metabolism in all vertebrates. T3 has both genomic and nongenomic effects on target cells. While much has been learnt on its genomic effects via T3 receptors (TRs) in vertebrate development, mostly through TR-knockout and TR-knockin studies, little is known about the effects of T3 on gene expression in animals in the absence of TR. We have been studying Xenopus metamorphosis as a model for mammalian postembryonic development, a period around birth when plasma T3 level peaks and many organs/tissues mature into their adult forms. We have recently generated TR double knockout (TRDKO) Xenopus tropicalis animals. This offers an opportunity to compare the effects of T3 on global gene expression in tadpole tissues in the presence or absence of TR. Methods: We analyzed the effects of T3 on gene expression in tadpole tail and intestine by using RNA-seq analysis on wild-type and TRDKO tadpoles with or without T3 treatment. Results: We observed that removing TRs reduced the number of genes regulated by T3 in both organs. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses revealed that T3 affected distinct biological processes and pathways in wild-type and TRDKO tadpoles. Many GO terms and KEGG pathways that were enriched among genes regulated in wild-type tissues are likely involved in mediating the effects of T3 on metamorphosis, for example, those related to development, stem cells, apoptosis, and cell cycle/cell proliferation. However, such GO terms and pathways were not enriched among T3-regulated genes in TRDKO tadpoles. Instead, in TRDKO tadpoles, GO terms and pathways related to "metabolism" and "immune response" were highly enriched among T3-regulated genes. We further observed strong divergence in the TR-independent nongenomic effects of T3 in the intestine and tail. Conclusions: Our data suggest that T3 has distinct and organ-dependent effects on gene expression in developing tadpoles. The TR-mediated effects are consistent with the metamorphic changes, in agreement with the fact that TR is necessary and sufficient to mediate the effects of T3 on metamorphosis. T3 appears to have a major effect on metabolism and immune response via TR-independent nongenomic processes.

Organ-specific effects on target binding due to knockout of thyroid hormone receptor α during Xenopus metamorphosis.

Thyroid hormone (T3) is essential for normal development and metabolism, especially during postembryonic development, a period around birth in mammals when plasma T3 levels reach their peak. T3 functions through two T3 receptors, TRα and TRß. However, little is known about the tissue-specific functions of TRs during postembryonic development because of maternal influence and difficulty in manipulation of mammalian models. We have studied Xenopus tropicalis metamorphosis as a model for human postembryonic development. By using TRα knockout (Xtr·thratmshi ) tadpoles, we have previously shown that TRα is important for T3-dependent intestinal remodeling and hindlimb development but not tail resorption during metamorphosis. Here, we have identified genes bound by TR in premetamorphic wild-type and Xtr·thratmshi tails with or without T3 treatment by using chromatin immunoprecipitation-sequencing and compared them with those in the intestine and hindlimb. Compared to other organs, the tail has much fewer genes bound by TR or affected by TRα knockout. Bioinformatic analyses revealed that among the genes bound by TR in wild-type but not Xtr·thratmshi organs, fewer gene ontology (GO) terms or biological pathways related to metamorphosis were enriched in the tail compared to those in the intestine and hindlimb. This difference likely underlies the drastic effects of TRα knockout on the metamorphosis of the intestine and hindlimb but not the tail. Thus, TRα has tissue-specific roles in regulating T3-dependent anuran metamorphosis by directly targeting the pathways and GO terms important for metamorphosis.

Citrus Huanglongbing correlated with incidence of Diaphorina citri carrying Candidatus Liberibacter asiaticus and citrus phyllosphere microbiome.

In China, citrus Huanglongbing (HLB) disease is caused by the Candidatus Liberibacter asiaticus bacterium, which is carried by the Asian citrus psyllid Diaphorina citri Kuwayama. It was hypothesized that the epidemic of the HLB may related with the rate of bacterium presence in the insect vector and bacterium content in plant tissues, as well as the phyllosphere microbe communities changes. This study systematically analyzed the presence or absence of Ca. L. asiaticus in citrus tree leaves and in the insect vector D. citri over a 6-year period using real-time PCR. In addition, changes in the number of bacteria carried by D. citri over 12 months were quantified, as well as the relationship between the proportion of D. citri carrying Ca. L. asiaticus and the proportion of plants infected with Ca. L. asiaticus were analyzed. Results showed that the proportion of D. citri carrying bacteria was stable and relatively low from January to September. The bacteria in citrus leaves relatively low in spring and summer, then peaked in December. The proportion of D. citri carrying bacteria gradually declined from 2014 to 2019. The proportion of D. citri carrying Ca. L. asiaticus showed a significant positive correlation with the proportion of diseased citrus. The phyllosphere bacterial and fungal communities on the healthy citrus leaf were significantly different with the disease leaf in April and December. Pathogenic invasions change the citrus phyllosphere microbial community structure. It could be summarized that citrus Huanglongbing correlated with incidence of Diaphorina citri carrying Candidatus Liberibacter asiaticus and citrus phyllosphere microbiome.

A 3' UTR-derived small RNA connecting nitrogen and carbon metabolism in enteric bacteria.

Increasing numbers of small, regulatory RNAs (sRNAs) corresponding to 3' untranslated regions (UTR) are being discovered in bacteria. One such sRNA, denoted GlnZ, corresponds to the 3' UTR of the Escherichia coli glnA mRNA encoding glutamine synthetase. Several forms of GlnZ, processed from the glnA mRNA, are detected in cells growing with limiting ammonium. GlnZ levels are regulated transcriptionally by the NtrC transcription factor and post-transcriptionally by RNase III. Consistent with the expression, E. coli cells lacking glnZ show delayed outgrowth from nitrogen starvation compared to wild type cells. Transcriptome-wide RNA-RNA interactome datasets indicated that GlnZ binds to multiple target RNAs. Immunoblots and assays of fusions confirmed GlnZ-mediated repression of glnP and sucA, encoding proteins that contribute to glutamine transport and the citric acid cycle, respectively. Although the overall sequences of GlnZ from E. coli K-12, Enterohemorrhagic E. coli and Salmonella enterica have significant differences due to various sequence insertions, all forms of the sRNA were able to regulate the two targets characterized. Together our data show that GlnZ impacts growth of E. coli under low nitrogen conditions by modulating genes that affect carbon and nitrogen flux.

Design, synthesis, and anticancer evaluation of ammosamide B with pyrroloquinoline derivatives as novel BRD4 inhibitors.

Bromodomain-containing protein 4 (BRD4), which is a member of the bromodomain and extra-terminal domain (BET) family, plays an important role in the regulation of gene expression as the "reader" of epigenetic regulation. BRD4 has become a promising target to treat cancer, because the up-regulation of BRD4 expression is closely associated with the occurrence and development of various cancers. At present, several BRD4 inhibitors are in clinical trials for cancer therapy, but no BRD4 inhibitors are on the market. Here, we designed and synthesized a series of compounds bearing pyrrolo[4,3,2-de]quinolin-2(1H)-one scaffold through structural modification of natural products ammosamide B, which is a natural pyrroloquinoline derivative reported for its potential antitumor activity. All target compounds were evaluated for their BRD4 BD1 inhibition activities via the protein thermal shift assays or AlphaSceen assay. The representative compound 49 showed potent activity (IC50 = 120 nM). The co-crystal of compound 49 with BRD4 BD1 was solved to study the structure activity relationship, which showed that 49 could combine with the acetyl lysine binding site and formed a hydrogen bond with the conserved residue Asn140. The results demonstrate that compound 49 is worthy of further investigation as a promising BRD4 inhibitor.

Thyroid hormone receptor α controls larval intestinal epithelial cell death by regulating the CDK1 pathway.

Thyroid hormone (T3) regulates adult intestine development through T3 receptors (TRs). It is difficult to study TR function during postembryonic intestinal maturation in mammals due to maternal influence. We chose intestinal remodeling during Xenopus tropicalis metamorphosis as a model to study TR function in adult organ development. By using ChIP (chromatin immunoprecipitation)-Seq, we identified over 3000 TR-bound genes in the intestine of premetamorphic wild type or TRα (the major TR expressed during premetamorphosis)-knockout tadpoles. Surprisingly, cell cycle-related GO (gene ontology) terms and biological pathways were highly enriched among TR target genes even though the first major event during intestinal metamorphosis is larval epithelial cell death, and TRα knockout drastically reduced this enrichment. More importantly, treatment of tadpoles with cell cycle inhibitors blocked T3-induced intestinal remodeling, especially larval epithelial cell death, suggesting that TRα-dependent activation of cell cycle is important for T3-induced apoptosis during intestinal remodeling.

Thyroid Hormone Receptor α Controls the Hind Limb Metamorphosis by Regulating Cell Proliferation and Wnt Signaling Pathways in Xenopus tropicalis.

Thyroid hormone (T3) receptors (TRs) mediate T3 effects on vertebrate development. We have studied Xenopus tropicalis metamorphosis as a model for postembryonic human development and demonstrated that TRα knockout induces precocious hind limb development. To reveal the molecular pathways regulated by TRα during limb development, we performed chromatin immunoprecipitation- and RNA-sequencing on the hind limb of premetamorphic wild type and TRα knockout tadpoles, and identified over 700 TR-bound genes upregulated by T3 treatment in wild type but not TRα knockout tadpoles. Interestingly, most of these genes were expressed at higher levels in the hind limb of premetamorphic TRα knockout tadpoles than stage-matched wild-type tadpoles, suggesting their derepression upon TRα knockout. Bioinformatic analyses revealed that these genes were highly enriched with cell cycle and Wingless/Integrated (Wnt) signaling-related genes. Furthermore, cell cycle and Wnt signaling pathways were also highly enriched among genes bound by TR in wild type but not TRα knockout hind limb. These findings suggest that direct binding of TRα to target genes related to cell cycle and Wnt pathways is important for limb development: first preventing precocious hind limb formation by repressing these pathways as unliganded TR before metamorphosis and later promoting hind limb development during metamorphosis by mediating T3 activation of these pathways.

Down-Regulation of Yeast Helicase Ded1 by Glucose Starvation or Heat-Shock Differentially Impairs Translation of Ded1-Dependent mRNAs.

Ded1 is an essential DEAD-box helicase in yeast that broadly stimulates translation initiation and is critical for mRNAs with structured 5'UTRs. Recent evidence suggests that the condensation of Ded1 in mRNA granules down-regulates Ded1 function during heat-shock and glucose starvation. We examined this hypothesis by determining the overlap between mRNAs whose relative translational efficiencies (TEs), as determined by ribosomal profiling, were diminished in either stressed WT cells or in ded1 mutants examined in non-stress conditions. Only subsets of the Ded1-hyperdependent mRNAs identified in ded1 mutant cells exhibited strong TE reductions in glucose-starved or heat-shocked WT cells, and those down-regulated by glucose starvation also exhibited hyper-dependence on initiation factor eIF4B, and to a lesser extent eIF4A, for efficient translation in non-stressed cells. These findings are consistent with recent proposals that the dissociation of Ded1 from mRNA 5'UTRs and the condensation of Ded1 contribute to reduced Ded1 function during stress, and they further suggest that the down-regulation of eIF4B and eIF4A functions also contributes to the translational impairment of a select group of Ded1 mRNA targets with heightened dependence on all three factors during glucose starvation.

Thyroid Hormone Receptor Is Essential for Larval Epithelial Apoptosis and Adult Epithelial Stem Cell Development but Not Adult Intestinal Morphogenesis during Xenopus tropicalis Metamorphosis.

Vertebrate postembryonic development is regulated by thyroid hormone (T3). Of particular interest is anuran metamorphosis, which offers several unique advantages for studying the role of T3 and its two nuclear receptor genes, TRα and TRß, during postembryonic development. We have recently generated TR double knockout (TRDKO) Xenopus tropicalis animals and reported that TR is essential for the completion of metamorphosis. Furthermore, TRDKO tadpoles are stalled at the climax of metamorphosis before eventual death. Here we show that TRDKO intestine lacked larval epithelial cell death and adult stem cell formation/proliferation during natural metamorphosis. Interestingly, TRDKO tadpole intestine had premature formation of adult-like epithelial folds and muscle development. In addition, T3 treatment of premetamorphic TRDKO tadpoles failed to induce any metamorphic changes in the intestine. Furthermore, RNA-seq analysis revealed that TRDKO altered the expression of many genes in biological pathways such as Wnt signaling and the cell cycle that likely underlay the inhibition of larval epithelial cell death and adult stem cell development caused by removing both TR genes. Our data suggest that liganded TR is required for larval epithelial cell degeneration and adult stem cell formation, whereas unliganded TR prevents precocious adult tissue morphogenesis such as smooth-muscle development and epithelial folding.

Reprogramming of translation in yeast cells impaired for ribosome recycling favors short, efficiently translated mRNAs.

In eukaryotes, 43S preinitiation complex (PIC) formation is a rate-determining step of translation. Ribosome recycling following translation termination produces free 40S subunits for re-assembly of 43S PICs. Yeast mutants lacking orthologs of mammalian eIF2D (Tma64), and either MCT-1 (Tma20) or DENR (Tma22), are broadly impaired for 40S recycling; however, it was unknown whether this defect alters the translational efficiencies (TEs) of particular mRNAs. Here, we conducted ribosome profiling of a yeast tma64∆/tma20∆ double mutant and observed a marked reprogramming of translation, wherein the TEs of the most efficiently translated ('strong') mRNAs increase, while those of 'weak' mRNAs generally decline. Remarkably, similar reprogramming was seen on reducing 43S PIC assembly by inducing phosphorylation of eIF2α or by decreasing total 40S subunit levels by depleting Rps26. Our findings suggest that strong mRNAs outcompete weak mRNAs in response to 43S PIC limitation achieved in various ways, in accordance with previous mathematical modeling.

Analysis of Thyroid Hormone Receptor α-Knockout Tadpoles Reveals That the Activation of Cell Cycle Program Is Involved in Thyroid Hormone-Induced Larval Epithelial Cell Death and Adult Intestinal Stem Cell Development During Xenopus tropicalis Metamorphosis.

Background: There are two highly conserved thyroid hormone (triiodothyronine [T3]) receptor (TR) genes, TRα and TRß, in all vertebrates, and the expression of TRα but not TRß is activated earlier than T3 synthesis during development. In human, high levels of T3 are present during the several months around birth, and T3 deficiency during this period causes severe developmental abnormalities including skeletal and intestinal defects. It is, however, difficult to study this period in mammals as the embryos and neonates depend on maternal supply of nutrients for survival. However, Xenopus tropicalis undergoes a T3-dependent metamorphosis, which drastically changes essentially every organ in a tadpole. Of interest is intestinal remodeling, which involves near complete degeneration of the larval epithelium through apoptosis. Concurrently, adult intestinal stem cells are formed de novo and subsequently give rise to the self-renewing adult epithelial system, resembling intestinal maturation around birth in mammals. We have previously demonstrated that T3 signaling is essential for the formation of adult intestinal stem cells during metamorphosis. Methods: We studied the function of endogenous TRα in the tadpole intestine by using knockout animals and RNA-seq analysis. Results: We observed that removing endogenous TRα caused defects in intestinal remodeling, including drastically reduced larval epithelial cell death and adult intestinal stem cell proliferation. Using RNA-seq on intestinal RNA from premetamorphic wild-type and TRα-knockout tadpoles treated with or without T3 for one day, before any detectable T3-induced cell death and stem cell formation in the tadpole intestine, we identified more than 1500 genes, which were regulated by T3 treatment of the wild-type but not TRα-knockout tadpoles. Gene Ontology and biological pathway analyses revealed that surprisingly, these TRα-regulated genes were highly enriched with cell cycle-related genes, in addition to genes related to stem cells and apoptosis. Conclusions: Our findings suggest that TRα-mediated T3 activation of the cell cycle program is involved in larval epithelial cell death and adult epithelial stem cell development during intestinal remodeling.

Dense Transposon Integration Reveals Essential Cleavage and Polyadenylation Factors Promote Heterochromatin Formation.

Heterochromatin functions as a scaffold for factors responsible for gene silencing and chromosome segregation. Heterochromatin can be assembled by multiple pathways, including RNAi and RNA surveillance. We identified factors that form heterochromatin using dense profiles of transposable element integration in Schizosaccharomyces pombe. The candidates include a large number of essential proteins such as four canonical mRNA cleavage and polyadenylation factors. We find that Iss1, a subunit of the poly(A) polymerase module, plays a role in forming heterochromatin in centromere repeats that is independent of RNAi. Genome-wide maps reveal that Iss1 accumulates at genes regulated by RNA surveillance. Iss1 interacts with RNA surveillance factors Mmi1 and Rrp6, and importantly, Iss1 contributes to RNA elimination that forms heterochromatin at meiosis genes. Our profile of transposable element integration supports the model that a network of mRNA cleavage and polyadenylation factors coordinates RNA surveillance, including the mechanism that forms heterochromatin at meiotic genes.

eIF1 discriminates against suboptimal initiation sites to prevent excessive uORF translation genome-wide.

The translation preinitiation complex (PIC) scans the mRNA for an AUG codon in a favorable context. Previous findings suggest that the factor eIF1 discriminates against non-AUG start codons by impeding full accommodation of Met-tRNAi in the P site of the 40S ribosomal subunit, necessitating eIF1 dissociation for start codon selection. Consistent with this, yeast eIF1 substitutions that weaken its binding to the PIC increase initiation at UUG codons on a mutant his4 mRNA and particular synthetic mRNA reporters; and also at the AUG start codon of the mRNA for eIF1 itself owing to its poor Kozak context. It was not known however whether such eIF1 mutants increase initiation at suboptimal start codons genome-wide. By ribosome profiling, we show that the eIF1-L96P variant confers increased translation of numerous upstream open reading frames (uORFs) initiating with either near-cognate codons (NCCs) or AUGs in poor context. The increased uORF translation is frequently associated with the reduced translation of the downstream main coding sequences (CDS). Initiation is also elevated at certain NCCs initiating amino-terminal extensions, including those that direct mitochondrial localization of the GRS1 and ALA1 products, and at a small set of main CDS AUG codons with especially poor context, including that of eIF1 itself. Thus, eIF1 acts throughout the yeast translatome to discriminate against NCC start codons and AUGs in poor context; and impairing this function enhances the repressive effects of uORFs on CDS translation and alters the ratios of protein isoforms translated from near-cognate versus AUG start codons.