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PubMed2002

The malaria parasite Plasmodium falciparum encodes members of the Puf RNA-binding protein family with conserved RNA binding activity.

A novel class of RNA-binding proteins, Puf, regulates translation and RNA stability by binding to specific sequences in the 3'-untranslated region of target mRNAs. Members of this protein family share a conserved Puf domain consisting of eight 36 amino acid imperfect repeats. Here we report two Puf family member genes, PfPuf1 and PfPuf2, from the human malaria parasite Plasmodium falciparum. Both genes are spliced with four and three introns clustered within or near the Puf domains, respectively. Northern and RT-PCR analysis indicated that both genes were differentially expressed in gametocytes during erythrocytic development of the parasite. Except for similarities in the Puf domain and expression profile, the deduced PfPuf1 and PfPuf2 proteins differ considerably in size and structure. PfPuf1 has 1894 amino acids and a central Puf domain, whereas PfPuf2 is much smaller with a C-terminal Puf domain. The presence of at least two Puf members in other Plasmodium species suggests that these proteins play evolutionarily similar roles during parasite development. Both in vivo studies using the yeast three-hybrid system and in vitro binding assays using the recombinant Puf domain of PfPuf1 expressed in bacteria demonstrated intrinsic binding activity of the PfPuf1 Puf domain to the NRE sequences in the hunchback RNA, the target sequence for Drosophila Pumilio protein. Altogether, these results suggest that PfPufs might function during sexual differentiation and development in Plasmodium through a conserved mechanism of translational regulation of their target mRNAs.

Amino Acid Sequence↗

PubMed2001

The rate of folate receptor alpha (FR alpha) synthesis in folate depleted CHL cells is regulated by a translational mechanism sensitive to media folate levels, while stable overexpression of its mRNA is mediated by gene amplification and an increase in transcript half-life.

DC-3F/FA3 cells (FA3) were obtained by selection of Chinese hamster lung fibroblasts for growth in folic acid free media, supplemented with 15 pM [6S]-5-formyltetrahydrofolic acid. These cells, as a result of low level gene amplification and RNA stabilization, were found to overexpress folate receptor alpha (FR alpha) mRNA by more than five hundred fold. The expression level of the receptor, a 43 kDa GPI-linked plasma membrane glycoprotein, was found to be inversely related to changes in media folate concentrations while its steady state mRNA level remained unaffected. In low folate, the rate of receptor synthesis was found to increase by more than three fold, while its half-life stabilized as compared to that observed in high folate media. Although DC-3F cells were found to contain low amounts of FR alpha mRNA, receptor expression was undetectable, and changing media folate concentrations had no effect on the expression of either. Hence, while selection for growth in low folate leads to stable overexpression of FR alpha mRNA, receptor expression is regulated at the level of protein synthesis by a mechanism sensitive to media folate levels.

Amino Acid Sequence↗

PubMed1998

IFN-gamma inhibits activation-induced expression of E- and P-selectin on endothelial cells.

E- and P-selectin are cell surface lectins that mediate leukocyte-endothelial cell adhesion and thereby participate in neutrophil recruitment into inflammatory sites. E-selectin can be induced on endothelial cells by various activators, including TNF-alpha, IL-1beta, and PMA. Induction of E-selectin is blocked by pretreatment of endothelial cells with IL-4 or TGF-beta, both of which have antiinflammatory properties in vivo. In addition to its well-known proinflammatory activities, IFN-gamma also has antiinflammatory effects in vivo, one of which is inhibition of neutrophil recruitment. To determine whether IFN-gamma inhibits neutrophil recruitment by inhibiting adhesion molecule expression, the effect of IFN-gamma on activation-induced cell adhesion molecule expression by cultured HUVEC was evaluated. Pretreatment of endothelial cells with IFN-gamma for 24 to 72 h before 6- to 24-h activation with IL-1beta, TNF-alpha, or PMA resulted in significantly reduced levels of cell surface E-selectin, although levels of ICAM-1 and VCAM-1 were the same or increased. The reduction of cell surface E-selectin levels under these conditions was reflected in reduced levels of E-selectin mRNA, indicating an effect at the transcription level or RNA stability. Interestingly, the increase of cell surface P-selectin expression due to IL-4 treatment of HUVEC was also inhibited by IFN-gamma, while constitutive levels of P-selectin were not. These results suggest that the inhibition of neutrophil recruitment by IFN-gamma in vivo may be due, in part, to the ability of IFN-gamma to inhibit E- and P-selectin up-regulation. Furthermore, these findings emphasize the process of leukocyte recruitment as an important step through which IFN-gamma can direct the character of inflammatory reactions.

Cell Adhesion↗

PubMed1997

Contributions of the procyclin 3' untranslated region and coding region to the regulation of expression in bloodstream forms of Trypanosoma brucei.

When bloodstream forms of Trypanosoma brucei differentiate into procyclic forms they rapidly synthesise a new surface coat composed of procyclins. Procyclin genes are transcribed in bloodstream forms at approximately one-tenth of the rate in procyclic forms, but little, if any, mRNA can be detected, indicating that further down-regulation must occur post-transcriptionally. We have examined the role of the 297 bp procyclin 3' untranslated region (UTR) in regulating expression in bloodstream forms and have identified three discrete elements: a dominant, negative element between positions 101 and 173, and two positive elements. When chloramphenicol acetyl transferase (CAT) was used as the reporter gene, deletion of the negative element caused a approximately 6-fold increase in the level of steady state mRNA and > 30-fold increase in CAT activity, suggesting that both RNA stability and translation were affected. Similar results were obtained with glutamic acid/alanine-rich protein (GARP), the T. congolense analogue of procyclin, indicating that the 3' UTR acts independently of the coding region. In contrast, when trypanosomes were stably transformed with a construct in which the procyclin coding region was linked to a truncated form of the 3' UTR which lacked the negative element, they expressed high levels of mRNA, but no protein could be detected in cell lysates or culture supernatants. These results imply that the procyclin coding region exerts yet another layer of control which prevents inappropriate expression of the protein in the mammalian host.

Amino Acid Sequence↗

PubMed2006

Analysis of SAGE data in human platelets: features of the transcriptome in an anucleate cell.

A comprehensive SAGE (serial analysis of gene expression) library of purified human platelets was established. Twenty-five thousand (25,000) tags were sequenced, and after removal of mitochondrial tags, 12,609 (51%) non-mitochondrial-derived tags remained, corresponding to 2,300 different transcripts with expression levels of up to 30,000 tags per million. This new, highly purified SAGE library of platelets is enriched in specific transcripts. The complexity in terms of tag distribution is similar to cells that are still able to replenish their mRNA pool by transcription. We show that our SAGE data are consistent with recently published microarray data but show further details of the platelet transcriptome, including (i) longer UTR regions and more stable folding in the enriched mRNAs, (ii) biologically interesting new candidate mRNAs that show regulatory elements, including elements for RNA stabilization or for translational control, and (iii) significant enrichment of specific, highly transcribed mRNAs compared to a battery of SAGE libraries from other tissues. Among several regulatory mRNA elements known to be involved in mRNA localization and translational control, CPE elements are in particular enriched in the platelet transcriptome. mRNAs previously reported to be translationally regulated were found to be present in the library and were validated by real-time PCR. Furthermore, specific molecular functions such as signal transduction activity were found to be significantly enriched in the platelet transcriptome. These findings emphasize the richness and diversity of the platelet transcriptome.

3' Untranslated Regions↗

PubMed1996

A suboptimal 5' splice site is a cis-acting determinant of nuclear export of polyomavirus late mRNAs.

Mouse polyomavirus has been used as a model system to study nucleocytoplasmic transport of mRNA. Three late mRNAs encoding the viral capsid proteins are generated by alternative splicing from common pre-mRNA molecules. mRNAs encoding the virion protein VP2 (mVP2) harbor an unused 5' splice site, and more than half of them remain fully unspliced yet are able to enter the cytoplasm for translation. Examination of the intracellular distribution of late viral mRNAs revealed, however, that mVP2 molecules are exported less efficiently than are mVP1 and mVP3, in which the 5' splice site has been removed by splicing. Point mutations and deletion analyses demonstrated that the efficiency of mVP2 export is inversely correlated with the strength of the 5' splice site and that unused 3' splice sites present in the mRNA have little or no effect on export. These results suggest that the unused 5' splice site is a key player in mVP2 export. Interestingly, mRNAs carrying large deletions but retaining the 5' splice site exhibited a wild-type mVP2 export phenotype, suggesting that there are no other constitutive cis-acting sequences involved in mVP2 export. RNA stability measurements confirmed that the subcellular distribution differences between these mRNAs were not due to differential half-lives between the two cellular compartments. We therefore conclude that the nuclear export of mVP2 is strongly influenced by a suboptimal 5' splice site. Furthermore, results comparing spliced and unspliced forms of mVP2 molecules indicated that the process of splicing does not enhance nuclear export. Since mVP2 and some of its mutant forms can accumulate in the cytoplasm in the absence of splicing, we propose that splicing is not a prerequisite for mRNA export in the polyomavirus system; rather, removal of splicing machinery from mRNAs may be required. The possibility that export of other viral mRNAs can be influenced by suboptimal splicing signals is also discussed.

3T3 Cells↗

PubMedDate not supplied

The multifaceted role of mTOR in cellular stress responses.

The mammalian target of rapamycin (mTOR) is a large multidomain protein whose function is inhibited by the immunosuppressant drug rapamycin. mTOR (or its homologues in lower eukaryotes) plays roles in cell growth and the cell cycle, control of the cytoskeleton and nutrient transport, protein and RNA stability and transcription and translation. In mammalian cells, the best understood effectors of mTOR are proteins involved in controlling the translational machinery. Signalling through mTOR is stimulated by amino acids and by hormones and mitogens. On the other hand, mTOR signaling is impaired in response to a range of stressful stimuli. These include DNA damage, nutrient withdrawal and depletion of cellular energy, as well as hypoxia. In response, e.g. to DNA damage, impairment of mTOR signaling appears to precede the commitment of cells to apoptosis. The mechanisms by which these stressful conditions still remain largely unclear. However, these responses make physiological sense, as impairment of mTOR signalling under these conditions will tend to inhibit anabolic processes and cell growth and division.

Animals↗

PubMed2026

Exploring genomic regions regulating the liver transcriptome and energy homeostasis in pigs.

In pigs, energy homeostasis has an impact on meat quality and health. In a Duroc pig population, 30 quantitative trait locus (QTL) regions associated with fatty acid (FA) composition in adipose tissue, plasma, liver and muscle were previously identified. Mapping of expression quantitative trait locus (eQTL) regions will provide a molecular hypothesis for genotype-phenotype interactions and may allow the identification of shared causal variants, key to increasing our understanding of the genetic regulation of FA composition and energy homeostasis. However, gene expression is impacted by environmental factors, while individual-level allelic imbalance (AI) can be more reliable and can be surveyed via allelic-specific expression (ASE) analysis. Furthermore, treatment of ASE as a quantitative trait allows the identification of allele-specific expression quantitative trait loci (aseQTLs), which are variants whose heterozygosity is linked to the AI of a nearby single-nucleotide polymorphism (SNP), pointing to regulatory elements. In this study, liver was selected as a key metabolic hub with an important role in the regulation of energy homeostasis, and 310 liver RNA sequencing samples were analysed using a combination of (1) eQTL mapping, (2) ASE analysis, and (3) aseQTL mapping methods. A total of 2 188 eQTL regions were identified, mostly cis-eQTL regions (73.17%). ASE analysis reported 1 964 ASE SNPs, associated with 633 genes. Finally, aseQTL mapping reported 64 172 aseQTL, associated with the AI of 31 genes. Colocalisation analysis combined with ASE analysis showed that the expression of FADS1 and FADS2 genes is associated with the polyunsaturated FA composition in several tissues, where microRNA regulation may be present. Finally, in the DGAT2 gene, annotated in a QTL region associated with multiple FAs in adipose tissue, ASE revealed allelic imbalance in the 3' untranslated region (UTR) of this gene. Allelic differential expression can be caused by a 13-bp insertion affecting messenger RNA stability, previously described, exemplifying how allelic imbalance is caused by a post-transcriptional regulatory mechanism undetectable by eQTL mapping. Furthermore, aseQTLs were associated with this gene, linked to a previously identified copy number variant not yet associated with DGAT2 expression. These results demonstrate how ASE analysis and aseQTL mapping can complement eQTL mapping, as they resolved a complex region affected by allelic heterogeneity, a main confounding effect of QTL mapping. In conclusion, the combination of eQTL mapping, ASE analysis and aseQTL mapping allowed the characterisation of the regulation of liver gene expression, improving our understanding of the genetic determinism of energy homeostasis.

Allele-specific expression↗

PubMed2000

Temporal control of protein synthesis during spermatogenesis.

During oogenesis and spermatogenesis transcription ceases prior to the differentiation of the mature cells. To complete germ cell differentiation and initiate early embryogenesis, proteins are synthesized from pre-existing mRNAs that are stored for several days. It is well established that important regulatory elements functioning in spatial localization, temporal translation or messenger RNA stability are located in the 3' untranslated region (3' UTR) of mRNAs. During mammalian spermatogenesis temporal translational regulation of the protamine 1 (Prm1) mRNA is dependent on a highly conserved sequence located in the distal region of its 3' UTR. The 17-nucleotide translational control element (TCE) mediates translational repression of the Prm1 mRNA. Mutation of the TCE causes premature synthesis of protamine protein and sterility. The Prm1 mRNA is stored as a cytoplasmic ribonucleoprotein (mRNP) particle in spermatids. Contained within the particle are several members of the Y box family of nucleic acid binding proteins. In the yeast three-hybrid system the murine Y box proteins MSY1, MSY2 and MSY4 bind in a sequence-dependent manner to a conserved region in the proximal portion of the Prm1 3' UTR. Sequence-specific binding by MSY4 to the Y box recognition sequence (YRS) is dependent on the highly conserved cold shock domain, possibly through the RNP1 and RNP2 motifs present within it. The Y box proteins may function as translational repressors in vivo. Alternatively, their primary function may be to protect mRNAs from degradation during their extended period of storage. Translational activation of stored mRNAs is essential for the completion of gametogenesis. Proper translational activation of the Prm1 mRNA in elongated spermatids requires the cytoplasmic double-stranded RNA binding protein TARBP2. Tarbp2 is expressed at low levels in many cells but is expressed at robust levels in late stage meiotic cells and in postmeiotic spermatids. Mice mutant for Tarbp2 are defective in proper translational activation of the Prm1 and Prm2 mRNAs and are sterile. Current studies are designed to determine the mechanism by which proteins bound to the 3' UTR communicate with the 5' end of the message to control translational silencing and activation.

Animals↗

PubMed2001

A 3'-transcribed region of the HLA-A2 gene mediates posttranscriptional stimulation by IFN-gamma.

The expression of several MHC class I genes is up-regulated at the transcriptional level by IFN-gamma. Posttranscriptional mechanisms also have been implicated, but not well characterized. To investigate the mechanism of IFN-gamma stimulation of the human MHC class I gene HLA-A2, several human tumor cell lines were transfected with reporter gene constructs driven by the HLA-A2 promoter. We have previously shown that the extended 525-bp HLA-A2 promoter alone, which includes a 5' IFN-stimulated response element consensus sequence, is not sufficient for IFN-gamma response in either K562 or Jurkat cells. In the current study, stable transfection of a genomic HLA-A2 gene construct, containing both 5'- and 3'-flanking sequences, resulted in stimulation of the gene by IFN-gamma. Nuclear run-on assays revealed that, unlike other class I genes, IFN-gamma stimulation of HLA-A mRNA accumulation occurs almost entirely through posttranscriptional mechanisms. RNA stability assays showed that the effect is not mediated by alteration of the half-life of the HLA-A2 mRNA. Formation of the 3' end was unaffected by IFN-gamma treatment. Sequences that mediate the majority of IFN-gamma induction of HLA-A2 mRNA reside in a 127-bp 3'-transcribed region of the gene. This region contains the terminal splice site, the usage of which is not affected by IFN-gamma treatment. These results demonstrate a novel posttranscriptional mechanism of regulation of MHC class I genes by IFN-gamma.

3' Untranslated Regions↗

PubMed1999

A premature termination codon interferes with the nuclear function of an exon splicing enhancer in an open reading frame-dependent manner.

Premature translation termination codon (PTC)-mediated effects on nuclear RNA processing have been shown to be associated with a number of human genetic diseases; however, how these PTCs mediate such effects in the nucleus is unclear. A PTC at nucleotide (nt) 2018 that lies adjacent to the 5' element of a bipartite exon splicing enhancer within the NS2-specific exon of minute virus of mice P4 promoter-generated pre-mRNA caused a decrease in the accumulated levels of P4-generated R2 mRNA relative to P4-generated R1 mRNA, although the total accumulated levels of P4 product remained the same. This effect was seen in nuclear RNA and was independent of RNA stability. The 5' and 3' elements of the bipartite NS2-specific exon enhancer are redundant in function, and when the 2018 PTC was combined with a deletion of the 3' enhancer element, the exon was skipped in the majority of the viral P4-generated product. Such exon skipping in response to a PTC, but not a missense mutation at nt 2018, could be suppressed by frame shift mutations in either exon of NS2 which reopened the NS2 open reading frame, as well as by improvement of the upstream intron 3' splice site. These results suggest that a PTC can interfere with the function of an exon splicing enhancer in an open reading frame-dependent manner and that the PTC is recognized in the nucleus.

Animals↗

PubMed1994

Developmental regulation of antisense-mediated gene silencing in Dictyostelium.

In Dictyostelium, the expression of antisense transcripts has been successfully used to reduce or eliminate gene expression. In most cases this occurs on the level of RNA stability resulting in a loss of both sense and antisense transcript accumulation. We here show that the antisense effect is regulated during the developmental cycle, i.e., in certain developmental stages and under certain developmental conditions, complementary RNAs appear not to interact with each other, resulting in a failure to abolish expression of the gene of interest. We find that this is not only the case with artificially introduced antisense constructs but also with the endogenous, antisense-regulated PSV-A gene. Our data demonstrate that antisense-mediated gene silencing is conferred by a biochemical machinery that is subject to regulation in vivo. The results provide a basis to better understand this machinery and to dissect the components. They may also explain the failure of some antisense experiments in Dictyostelium and possibly in other organisms.

Animals↗

PubMed2004

Effects of low density lipoprotein receptor-related protein-1 on the expression of platelet-derived growth factor beta-receptor in vitro.

The low density lipoprotein receptor related protein-1 (LRP-1) is a cargo transport receptor that undergoes constitutive endocytosis and recycling. Platelet-derived growth factor-BB (PDGF-BB) binds to LRP-1 and may bridge LRP-1 to PDGF receptors. Bridging of LRP-1 to other receptors by bifunctional ligands may represent a general mechanism whereby LRP-1 facilitates internalization of membrane proteins. The goal of this study was to determine whether LRP-1 regulates cell-surface levels of PDGF beta-receptor or PDGF beta-receptor degradation following treatment with PDGF-BB. Unexpectedly, in both murine embryonic fibroblasts (MEFs) and HT 1080 fibrosarcoma cells, LRP-1 expression was associated with increased levels of PDGF beta-receptor. In MEFs, the mechanism involved increased PDGF beta-receptor transcription and/or RNA stabilization. LRP-1 expression was not associated with increased levels of PDGF beta-receptor in Chinese hamster ovary (CHO) cells, suggesting that cell context is important. The kinetics of PDGF beta-receptor phosphorylation, in response to PDGF-BB, and the extent of degradation of PDGF beta-receptor were equivalent in LRP-1-expressing and -deficient MEFs. We conclude that PDGF beta-receptor expression and cell surface levels may be regulated by LRP-1; however, this activity is cell type-specific. LRP-1 does not directly regulate PDGF beta-receptor phosphorylation or degradation in PDGF-BB-treated cells.

Animals↗

PubMed1993

Expression and regulation of human neutrophil-derived macrophage inflammatory protein 1 alpha.

Neutrophil (polymorphonuclear leukocyte [PMN]) sequestration is one of the histologic hallmarks of an acute inflammatory response. During the natural evolution of an inflammatory response, PMNs are often replaced by mononuclear cells. This shift in the elicitation of specific leukocyte populations usually occurs as the inflammatory lesion enters either the repair/resolution stage or progresses to a chronic inflammation. To elucidate a potential mechanism for the temporal change from predominantly PMN recruitment to the presence of monocytes, we postulated that PMNs could be a rich source of monocyte chemotactic factors. In our studies, we have identified a dose-dependent induction of monocyte chemotactic activity by PMNs treated with lipopolysaccharide (LPS; 1-100 ng/ml). Interestingly, this monocyte chemotactic activity was significantly attenuated in the presence of neutralizing anti-human macrophage inflammatory protein 1 alpha (MIP-1 alpha) antibodies. Moreover, immunolocalization studies demonstrated the expression of MIP-1 alpha by stimulated PMNs. These findings showed that a significant amount of PMN-derived monocyte chemotactic activity was attributable to MIP-1 alpha. Subsequent characterization of MIP-1 alpha steady-state mRNA and antigen expression demonstrated both a dose- and time-dependent production by LPS-treated PMNs. Granulocyte/macrophage colony-stimulating factor (GM-CSF), a potent PMN activator, failed to induce the expression of MIP-1 alpha over a wide range of concentrations. However, PMNs stimulated in the presence of both LPS and GM-CSF resulted in a synergistic expression pattern for MIP-1 alpha. PMNs stimulated in the presence of both GM-CSF and LPS demonstrated an enhanced and prolonged expression for both MIP-1 alpha mRNA and antigen, as compared with LPS alone. Messenger RNA stabilization analyses demonstrated that MIP-1 alpha mRNA isolated from PMNs stimulated in the presence of GM-CSF and LPS had a prolonged mRNA t1/2, as compared with LPS alone. These findings support the notion that PMNs are capable of producing MIP-1 alpha in the presence of LPS, and that GM-CSF can influence this production through prolongation of MIP-1 alpha mRNA t1/2. The production of PMN-derived MIP-1 alpha, in association with the expression of appropriate adhesion molecules at a site of inflammation, may be one of the central events that contributes to the temporal shift from predominantly PMNs to monocytes during the evolution of inflammation.

Base Sequence↗

PubMed2006

Derepression of the Her-2 uORF is mediated by a novel post-transcriptional control mechanism in cancer cells.

Transcripts harboring 5' upstream open reading frames (uORFs) are often found in genes controlling cell growth including receptors, oncogenes, or growth factors. uORFs can modulate translation or RNA stability and mediate inefficient translation of these potent proteins under normal conditions. In dysregulated cancer cells, where the gene product, for example Her-2 receptor, is overexpressed, post-transcriptional processes must exist that serve to override the inhibitory effects of the uORFs. The 5' untranslated region (UTR) of Her-2 mRNA contains a short uORF that represses translation of the downstream coding region. We demonstrate that in Her-2 overexpressing breast cancer cells, the 3' UTR of the Her-2 mRNA can override translational inhibition mediated by the Her-2 uORF. Within this 3' UTR, a translational derepression element (TDE) that binds to a 38-kDa protein was identified. These results define a novel biological mechanism in which translational control of genes harboring a 5' uORF can be modulated by elements in their 3' UTRs.

Base Sequence↗

PubMed2005

Allelic effects on gene regulation at the autoimmunity-predisposing CTLA4 locus: a re-evaluation of the 3' +6230G>A polymorphism.

Genetic variation at a linkage disequilibrium block encompassing the cytotoxic T-lymphocyte antigen-4 (CTLA4) gene influences susceptibility to autoimmunity, but identifying the polymorphism(s) responsible for this effect has been challenging. Recently, a single-nucleotide polymorphism (SNP) located 3' to the known polyadenylation site of CTLA4 (+6230G>A) and strongly associated with autoimmune disease was reported to regulate levels of soluble CTLA4 isoform (sCTLA4) but not the full-length isoform. The purpose of the present study is to define the mechanistic effect of the 3'SNP on the isoforms of CTLA4 (alternative splicing vs polyadenylation vs effects on RNA stability). However, using allele-specific single-nucleotide primer extension, we found no difference between mRNA transcripts derived from either +6230G>A allele in 11 heterozygous individuals, in either of the two known CTLA4 isoforms. We also found no effect of this polymorphism on ICOS (inducible costimulator), a putative downstream target. In addition, repeated attempts at 3' RACE (3'rapid amplification of cDNA ends) were unsuccessful in amplifying any contiguous sequence past the known CTLA4 polyadenylation site and no such sequence was found in the EST databases. We conclude that the mechanism of the observed association of the +6230 SNP with autoimmune disease remains to be determined, but does not involve modulation of steady-state mRNA of any known CTLA4 isoform.

3' Flanking Region↗

PubMed2025

An RNA polymerase III tissue and tumor atlas uncovers context-specific activities linked to 3D epigenome regulatory mechanisms.

RNA polymerase III (Pol III) produces a plethora of small noncoding RNA species involved in diverse cellular processes, from transcription regulation and splicing to RNA stability, translation, and proteostasis. Though Pol III activity is broadly coupled with cellular demands for protein synthesis and growth, a more precise understanding of gene-level dynamics and context-specific expression patterns remains missing, in part due to challenges related to sequencing and mapping Pol III-derived small ncRNAs. Here, we establish a predictive multi-tissue map of human Pol III activity across 19 tissues and 23 primary cancer subtypes by comprehensively profiling the chromatin accessibility of canonical Pol III-transcribed gene classes. Our framework relies on the unique relationship between gene accessibility and Pol III transcription, inferring activity through uniform binary classification of ATAC-seq enrichment at Pol III-transcribed genes. By characterizing multi-context gene uniformity, we provide a definition of the core Pol III transcriptome, broadly active across specialized tissues, and catalog genes with varied levels of context specificity. Our genomic Pol III atlas uncovers variable levels of activity across tissues, including sharp contraction of the Pol III transcriptome in heart and brain tissues and frequent expansion across diverse cancers. We show that both tissue- and tumor-specific genes are significantly enriched within lamina-associated domains (LADs), and that aberrant expression of nuclear lamin proteins is sufficient to induce Pol III-emergent patterns at tumor-specific genes. Together, these findings link Pol III dynamics to subnuclear compartmentalization and provide a resource for better understanding Pol III expansion and small RNA biogenesis in cancer.

Journal Article↗

PubMed2001

Increased expression of alpha 1A Ca2+ channel currents arising from expanded trinucleotide repeats in spinocerebellar ataxia type 6.

The expansion of polyglutamine tracts encoded by CAG trinucleotide repeats is a common mutational mechanism in inherited neurodegenerative diseases. Spinocerebellar ataxia type 6 (SCA6), an autosomal dominant, progressive disease, arises from trinucleotide repeat expansions present in the coding region of CACNA1A (chromosome 19p13). This gene encodes alpha(1A), the principal subunit of P/Q-type Ca(2+) channels, which are abundant in the CNS, particularly in cerebellar Purkinje and granule neurons. We assayed ion channel function by introduction of human alpha(1A) cDNAs in human embryonic kidney 293 cells that stably coexpressed beta(1) and alpha(2)delta subunits. Immunocytochemical analysis showed a rise in intracellular and surface expression of alpha(1A) protein when CAG repeat lengths reached or exceeded the pathogenic range for SCA6. This gain at the protein level was not a consequence of changes in RNA stability, as indicated by Northern blot analysis. The electrophysiological behavior of alpha(1A) subunits containing expanded (EXP) numbers of CAG repeats (23, 27, and 72) was compared against that of wild-type subunits (WT) (4 and 11 repeats) using standard whole-cell patch-clamp recording conditions. The EXP alpha(1A) subunits yielded functional ion channels that supported inward Ca(2+) channel currents, with a sharp increase in P/Q Ca(2+) channel current density relative to WT. Our results showed that Ca(2+) channels from SCA6 patients display near-normal biophysical properties but increased current density attributable to elevated protein expression at the cell surface.

Blotting, Northern↗