Genes In Higher Eukaryotes Research Articles

Diverse cell-specific patterns of alternative polyadenylation in Drosophila

Most genes in higher eukaryotes express isoforms with distinct 3’ untranslated regions (3’ UTRs), generated by alternative polyadenylation (APA). Since 3’ UTRs are predominant locations of post-transcriptional regulation, APA can render such programs conditional, and can also alter protein sequences via alternative last exon (ALE) isoforms. We previously used 3’-sequencing from diverse Drosophila samples to define multiple tissue-specific APA landscapes. Here, we exploit comprehensive single nucleus RNA-sequencing data (Fly Cell Atlas) to elucidate cell-type expression of 3’ UTRs across >250 adult Drosophila cell types. We reveal the cellular bases of multiple tissue-specific APA/ALE programs, such as 3’ UTR lengthening in differentiated neurons and 3’ UTR shortening in spermatocytes and spermatids. We trace dynamic 3’ UTR patterns across cell lineages, including in the male germline, and discover new APA patterns in the intestinal stem cell lineage. Finally, we correlate expression of RNA binding proteins (RBPs), miRNAs and global levels of cleavage and polyadenylation (CPA) factors in several cell types that exhibit characteristic APA landscapes, yielding candidate regulators of transcriptome complexity. These analyses provide a comprehensive foundation for future investigations of mechanisms and biological impacts of alternative 3’ isoforms across the major cell types of this widely-studied model organism.

BET inhibition disrupts transcription but retains enhancer-promoter contact

Enhancers are DNA sequences that enable complex temporal and tissue-specific regulation of genes in higher eukaryotes. Although it is not entirely clear how enhancer-promoter interactions can increase gene expression, this proximity has been observed in multiple systems at multiple loci and is thought to be essential for the maintenance of gene expression. Bromodomain and Extra-Terminal domain (BET) and Mediator proteins have been shown capable of forming phase condensates and are thought to be essential for super-enhancer function. Here, we show that targeting of cells with inhibitors of BET proteins or pharmacological degradation of BET protein Bromodomain-containing protein 4 (BRD4) has a strong impact on transcription but very little impact on enhancer-promoter interactions. Dissolving phase condensates reduces BRD4 and Mediator binding at enhancers and can also strongly affect gene transcription, without disrupting enhancer-promoter interactions. These results suggest that activation of transcription and maintenance of enhancer-promoter interactions are separable events. Our findings further indicate that enhancer-promoter interactions are not dependent on high levels of BRD4 and Mediator, and are likely maintained by a complex set of factors including additional activator complexes and, at some sites, CTCF and cohesin.

Enhancing transient protein expression in HEK-293 cells by briefly exposing the culture to DMSO

BackgroundTransient expression of proteins in mammalian cells is a key technique for many functional and structural studies of human and higher eukaryotic genes as well as for the production of recombinant protein therapeutics. Maximizing the expression efficiency to achieve a higher expression yield is desirable and may be even critical when, for instance, an expressed protein must be characterized at the single-cell level. New MethodsOur goal was to develop a simple method by which protein expression yield in human embryonic kidney (HEK)-293 cells could be enhanced with a brief treatment of dimethyl sulfoxide (DMSO) solution. ResultsBy expressing green fluorescent protein (GFP) as a reporter protein using the calcium phosphate transfection method and imaging a large population of cells, we found that a 5-min exposure of 10 % DMSO to HEK-293 cells, 4 h after transfection of the protein of interest, leads to ∼1.6-fold increase in the expression yield without causing any appreciable cytotoxicity. By expressing an α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and separately a kainate receptor in HEK-293 cells and measuring glutamate-induced whole-cell current response, we also found that such a brief DMSO treatment did not affect channel activity. ConclusionThis method is simple, efficient and inexpensive to use for enhancing transient transfection yield in HEK-293 cells.

JMJD5 couples with CDK9 to release the paused RNA polymerase II

More than 30% of genes in higher eukaryotes are regulated by RNA polymerase II (Pol II) promoter proximal pausing. Pausing is released by the positive transcription elongation factor complex (P-TEFb). However, the exact mechanism by which this occurs and whether phosphorylation of the carboxyl-terminal domain of Pol II is involved in the process remains unknown. We previously reported that JMJD5 could generate tailless nucleosomes at position +1 from transcription start sites (TSS), thus perhaps enable progression of Pol II. Here we find that knockout of JMJD5 leads to accumulation of nucleosomes at position +1. Absence of JMJD5 also results in loss of or lowered transcription of a large number of genes. Interestingly, we found that phosphorylation, by CDK9, of Ser2 within two neighboring heptad repeats in the carboxyl-terminal domain of Pol II, together with phosphorylation of Ser5 within the second repeat, HR-Ser2p (1, 2)-Ser5p (2) for short, allows Pol II to bind JMJD5 via engagement of the N-terminal domain of JMJD5. We suggest that these events bring JMJD5 near the nucleosome at position +1, thus allowing JMJD5 to clip histones on this nucleosome, a phenomenon that may contribute to release of Pol II pausing.

The expanding regulatory mechanisms and cellular functions of circular RNAs.

Many protein-coding genes in higher eukaryotes can produce circular RNAs (circRNAs) through back-splicing of exons. CircRNAs differ from mRNAs in their production, structure and turnover and thereby have unique cellular functions and potential biomedical applications. In this Review, I discuss recent progress in our understanding of the biogenesis of circRNAs and the regulation of their abundance and of their biological functions, including in transcription and splicing, sequestering or scaffolding of macromolecules to interfere with microRNA activities or signalling pathways, and serving as templates for translation. I further discuss the emerging roles of circRNAs in regulating immune responses and cell proliferation, and the possibilities of applying circRNA technologies in biomedical research.

JMJD6 cleaves MePCE to release positive transcription elongation factor b (P-TEFb) in higher eukaryotes.

More than 30% of genes in higher eukaryotes are regulated by promoter-proximal pausing of RNA polymerase II (Pol II). Phosphorylation of Pol II CTD by positive transcription elongation factor b (P-TEFb) is a necessary precursor event that enables productive transcription elongation. The exact mechanism on how the sequestered P-TEFb is released from the 7SK snRNP complex and recruited to Pol II CTD remains unknown. In this report, we utilize mouse and human models to reveal methylphosphate capping enzyme (MePCE), a core component of the 7SK snRNP complex, as the cognate substrate for Jumonji domain-containing 6 (JMJD6)'s novel proteolytic function. Our evidences consist of a crystal structure of JMJD6 bound to methyl-arginine, enzymatic assays of JMJD6 cleaving MePCE in vivo and in vitro, binding assays, and downstream effects of Jmjd6 knockout and overexpression on Pol II CTD phosphorylation. We propose that JMJD6 assists bromodomain containing 4 (BRD4) to recruit P-TEFb to Pol II CTD by disrupting the 7SK snRNP complex.

Newly acquired N-terminal extension targets threonyl-tRNA synthetase-like protein into the multiple tRNA synthetase complex.

A typical feature of eukaryotic aminoacyl-tRNA synthetases (aaRSs) is the evolutionary gain of domains at either the N- or C-terminus, which frequently mediating protein–protein interaction. TARSL2 (mouse Tarsl2), encoding a threonyl-tRNA synthetase-like protein (ThrRS-L), is a recently identified aaRS-duplicated gene in higher eukaryotes, with canonical functions in vitro, which exhibits a different N-terminal extension (N-extension) from TARS (encoding ThrRS). We found the first half of the N-extension of human ThrRS-L (hThrRS-L) is homologous to that of human arginyl-tRNA synthetase. Using the N-extension as a probe in a yeast two-hybrid screening, AIMP1/p43 was identified as an interactor with hThrRS-L. We showed that ThrRS-L is a novel component of the mammalian multiple tRNA synthetase complex (MSC), and is reliant on two leucine zippers in the N-extension for MSC-incorporation in humans, and mouse cell lines and muscle tissue. The N-extension was sufficient to target a foreign protein into the MSC. The results from a Tarsl2-deleted cell line showed that it does not mediate MSC integrity. The effect of phosphorylation at various sites of hThrRS-L on its MSC-targeting is also explored. In summary, we revealed that ThrRS-L is a bona fide component of the MSC, which is mediated by a newly evolved N-extension domain.

Rice Trithorax factor ULTRAPETALA 1 (OsULT1) specifically binds to “GAGAG” sequence motif present in Polycomb response elements

Co-ordinated interplay between Polycomb group (PcG) and Trithorax group (TrxG) of proteins regulate chromatin state and maintain the transcription “off” and “on” state of a gene in higher eukaryotes. Targeting PcG complex to a specific locus is mediated by DNA sequences known as Polycomb response elements. Interestingly, these PREs are also recognized by TrxG proteins to antagonise PcG mediated gene repression. In this study, we have characterised DNA binding property of rice trithorax group factor ULTRAPETALA1 (OsULT1) which has a SAND domain and B–box motif. Chromatin immunoprecipitation assay indicates cold induced enrichment of OsULT1 occupancy and a decrease in H3K27me3 mark in the promoter region of OsDREB1b gene, during transcription activation. OsULT1 binds to the cis motif “GAGAG”, and the sequence specificity is contributed mainly by the SAND domain. GAGAG is one of the cis motifs present in PREs that are recognized by Drosophila GAGA factor and Pipsqueak. Thus, binding of OsULT1 to GAGAG motif, along with a decrease in H3K27me3 suggests that OsULT1 antagonises the repressive effect of PcG complex for transcriptional activation of OsDREB1b. Moreover, OsULT1 interacts with rice SET domain-containing methyltransferase TRX1, suggesting OsULT1 is an integral part of plant Trithorax group complex. Furthermore, the increase in ULT1 levels during environmental cues suggests its involvement in the transcriptional regulation of stress responsive genes. Collectively, these results suggest that the antagonistic functions of PcG and TrxG proteins and the mechanism of recruitment of these complexes to target loci are evolutionarily conserved for gene expression regulation across kingdoms.

Diversification of DNA binding specificities enabled SREBP transcription regulators to expand the repertoire of cellular functions that they govern in fungi.

The Sterol Regulatory Element Binding Proteins (SREBPs) are basic-helix-loop-helix transcription regulators that control the expression of sterol biosynthesis genes in higher eukaryotes and some fungi. Surprisingly, SREBPs do not regulate sterol biosynthesis in the ascomycete yeasts (Saccharomycotina) as this role was handed off to an unrelated transcription regulator in this clade. The SREBPs, nonetheless, expanded in fungi such as the ascomycete yeasts Candida spp., raising questions about their role and evolution in these organisms. Here we report that the fungal SREBPs diversified their DNA binding preferences concomitantly with an expansion in function. We establish that several branches of fungal SREBPs preferentially bind non-palindromic DNA sequences, in contrast to the palindromic DNA motifs recognized by most basic-helix-loop-helix proteins (including SREBPs) in higher eukaryotes. Reconstruction and biochemical characterization of the likely ancestor protein suggest that an intrinsic DNA binding promiscuity in the family was resolved by alternative mechanisms in different branches of fungal SREBPs. Furthermore, we show that two SREBPs in the human commensal yeast Candida albicans drive a transcriptional cascade that inhibits a morphological switch under anaerobic conditions. Preventing this morphological transition enhances C. albicans colonization of the mammalian intestine, the fungus’ natural niche. Thus, our results illustrate how diversification in DNA binding preferences enabled the functional expansion of a family of eukaryotic transcription regulators.

Stochastic Gene Choice during Cellular Differentiation

Genes in higher eukaryotes are regulated by long-range interactions, which can determine what combination of genes is expressed in a chromosomal segment. The choice of the genes can display exclusivity, independence, or co-occurrence. We introduced a simple measure to quantify this interdependence in gene expression and differentiated mouse embryonic stem cells to neurons to measure the single-cell expression of the gene isoforms in the protocadherin (Pcdh) cluster, a key component of neuronal diversity. As the neuronal progenitors mature into neurons, expression of the gene isoforms in the Pcdh array is initially concurrent. Even though the number of the expressed genes is increasing during differentiation, the expression shifts toward exclusivity. The expression frequency correlates highly with CTCF binding to the promoters and follows dynamically the changes in the binding during the differentiation. These findings aid in understanding the interplay between cellular differentiation and stochastic gene choice.

Abstract IA13: Ribosome stalling and disease

Abstract La Jolla, CA. Protein synthesis is vital for cell function and disturbances in translation are associated with cancer, neurological disorders and other diseases. Although regulation of translation is thought to largely occur at initiation, elongation, the reiterative addition of amino acids by the ribosome, is emerging as a translational phase that is also highly regulated. Transfer RNAs, the adaptor molecules essential for elongation are encoded by several hundreds of nuclear genes in higher eukaryotes suggesting great functional redundancy. Our work has defined epistatic interactions between mutations in a mammalian tRNA gene and a novel ribosome rescue factor, defining the importance of individual tRNA genes in translation and the pathologies that can arise from disruptions in this process. Citation Format: Susan L. Ackerman. Ribosome stalling and disease. [abstract]. In: Proceedings of the AACR Special Conference on Translational Control of Cancer: A New Frontier in Cancer Biology and Therapy; 2016 Oct 27-30; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2017;77(6 Suppl):Abstract nr IA13.

Transcription factors AS1 and AS2 interact with LHP1 to repress KNOX genes in Arabidopsis.

Polycomb group proteins are important repressors of numerous genes in higher eukaryotes. However, the mechanism by which Polycomb group proteins are recruited to specific genes is poorly understood. In Arabidopsis, LIKE HETEROCHROMATIN PROTEIN 1 (LHP1), also known as TERMINAL FLOWER 2, was originally proposed as a subunit of polycomb repressive complex 1 (PRC1) that could bind the tri-methylated lysine 27 of histone H3 (H3K27me3) established by the PRC2. In this work, we show that LHP1 mainly functions with PRC2 to establish H3K27me3, but not with PRC1 to catalyze monoubiquitination at lysine 119 of histone H2A. Our results show that complexes of the transcription factors ASYMMETRIC LEAVES 1 (AS1) and AS2 could help to establish the H3K27me3 modification at the chromatin regions of Class-I KNOTTED1-like homeobox (KNOX) genes BREVIPEDICELLUS and KNAT2 via direct interactions with LHP1. Additionally, our transcriptome analysis indicated that there are probably more common target genes of AS1 and LHP1 besides Class-I KNOX genes during leaf development in Arabidopsis.

Fundamentals of Expression in Mammalian Cells.

Expression of proteins in mammalian cells is a key technology important for many functional studies on human and higher eukaryotic genes. Studies include the mapping of protein interactions, solving protein structure by crystallization and X-ray diffraction or solution phase NMR and the generation of antibodies to enable a range of studies to be performed including protein detection in vivo. In addition the production of therapeutic proteins and antibodies, now a multi billion dollar industry, has driven major advances in cell line engineering for the production of grams per liter of active proteins and antibodies. Here the key factors that need to be considered for successful expression in HEK293 and CHO cells are reviewed including host cells, expression vector design, transient transfection methods, stable cell line generation and cultivation conditions.

Identification and Correction of Erroneous Protein Sequences in Public Databases.

Correct prediction of the structure of protein-coding genes of higher eukaryotes is a difficult task therefore public sequence databases incorporating predicted sequences are increasingly contaminated with erroneous sequences. The high rate of misprediction has serious consequences since it significantly affects the conclusions that may be drawn from genome-scale sequence analyses.Here we describe the MisPred and FixPred approaches that may help the identification and correction of erroneous sequences. The rationale of these approaches is that a protein sequence is likely to be erroneous if some of its features conflict with our current knowledge about proteins.

RNA and lessons from viruses.

RNA and lessons from viruses.

RADPRE: a computational program for identification of differential mRNA processing including alternative polyadenylation.

Genome-wide studies revealed the prevalence of multiple transcripts resulting from alternative polyadenylation (APA) of a single given gene in higher eukaryotes. Several studies in the past few years attempted to address how those APA events are regulated and what the biological consequences of those regulations are. Common to these efforts is the comparison of unbiased transcriptome data, either derived from whole-genome tiling array or next generation sequencing, to identify the specific APA events in a given condition. RADPRE (Ratio-based Analysis of Differential mRNA Processing and Expression) is an R program, developed to serve such a purpose using data from the whole-genome tilling array. RADPRE took a set of tilling array data as input, performed a series of calculation including a correction of the probe affinity variation, a hierarchy of statistical tests and an estimation of the false discovery rate (FDR) of the differentially processed genes (DPG). The result was an output of a few tabular files including DPG and their corresponding FDR. This chapter is written for scientists with limited programming experiences.

Structural biology: Enzyme-chromatin complex visualized.

The structure of an enzyme that is bound to a nucleosome — a protein complex around which DNA is wrapped — reveals how contacts between the two orient the enzyme so that it can modify a specific amino-acid residue. See Article p.591 Polycomb group proteins repress the expression of developmentally regulated genes in higher eukaryotes. Polycomb repressive complex 1 (PRC1) ubiquitylates histone H2A using E3 ubiquitin ligase subunits, Ring1B and Bmi1, together with an E2 ubiquitin-conjugating enzyme, UbcH5c. Here, Song Tan and colleagues describe the crystal structure of the PRC1 ubiquitylation module bound to its nucleosome core substrate. The structure shows how a histone modifying enzyme achieves substrate specificity by interacting with multiple nucleosome surfaces spatially distinct from the site of catalysis, and reveals an unexpected role for the E2 enzyme in substrate recognition. Based on their new data, the authors generate a model for nucleosome binding of the related BRCA1 H2A E3 ligase that can be used to examine the role of BRCA1's histone-modifying activity in development and cancer predisposition.

Splicing together sister chromatids.

Splicing of pre‐mRNAs is a necessary step for expression of the majority of genes in higher eukaryotes, and its regulation through alternative splice site selection shapes their proteomes. Defects in multiple splicing factors result in aberrant mitotic progression, although the molecular basis for this observation has remained elusive. Recent papers in The EMBO Journal and EMBO Reports reveal that expression of sororin, a critical regulator that stabilizes cohesin rings in sister chromatids, is exquisitely sensitive to defects in the splicing machinery, thus explaining the striking link between spliceosome function and chromosome segregation.

PrimerSeq: Design and Visualization of RT-PCR Primers for Alternative Splicing Using RNA-seq Data

The vast majority of multi-exon genes in higher eukaryotes are alternatively spliced and changes in alternative splicing (AS) can impact gene function or cause disease. High-throughput RNA sequencing (RNA-seq) has become a powerful technology for transcriptome-wide analysis of AS, but RT-PCR still remains the gold-standard approach for quantifying and validating exon splicing levels. We have developed PrimerSeq, a user-friendly software for systematic design and visualization of RT-PCR primers using RNA-seq data. PrimerSeq incorporates user-provided transcriptome profiles (i.e., RNA-seq data) in the design process, and is particularly useful for large-scale quantitative analysis of AS events discovered from RNA-seq experiments. PrimerSeq features a graphical user interface (GUI) that displays the RNA-seq data juxtaposed with the expected RT-PCR results. To enable primer design and visualization on user-provided RNA-seq data and transcript annotations, we have developed PrimerSeq as a stand-alone software that runs on local computers. PrimerSeq is freely available for Windows and Mac OS X along with source code at http://primerseq.sourceforge.net/. With the growing popularity of RNA-seq for transcriptome studies, we expect PrimerSeq to help bridge the gap between high-throughput RNA-seq discovery of AS events and molecular analysis of candidate events by RT-PCR.

The ribosomal protein rpl26 promoter is required for its 3′ sense terminus ncRNA transcription in Schizosaccharomyces pombe, implicating a new transcriptional mechanism for ncRNAs

Transcriptome studies have revealed that many non-coding RNAs (ncRNAs) are located near the 3′ sense terminus of protein-coding genes. However, the transcription and function of these RNAs remain elusive. Here, we identify a 3′ sense termini-associated sRNA (TASR) downstream of rpl26 in Schizosaccharomyces pombe (S. pombe). Structure and function assays indicate that the TASR is an H/ACA box snoRNA required for 18S rRNA pseudouridylation at U121 and U305 sites and is therefore a cognate of snR49 from the budding yeast. Transcriptional studies show that pre-snR49 overlaps most of the coding sequence (CDS) of rpl26. Using scanning deletion analysis within promoter region, we show that the rpl26 promoter is required for the 3′ TASR transcription. Interestingly, chromosomal synteny of rpl26–snR49 is found in the Schizosaccharomyces groups. Taken together, we have revealed a new transcriptional mechanism for 3′ sense TASRs, which are transcribed by the same promoter as their upstream protein genes. These results further suggest that the origin and function of 3′ sense ncRNAs are associated with upstream genes in higher eukaryotes.