End Formation Research Articles

The CBC social network

The formation of diverse CBC subcomplexes determines RNA fate by connecting 3′ end formation, degradation and export.

Transcription Termination and Chimeric RNA Formation Controlled by Arabidopsis thaliana FPA

Alternative cleavage and polyadenylation influence the coding and regulatory potential of mRNAs and where transcription termination occurs. Although widespread, few regulators of this process are known. The Arabidopsis thaliana protein FPA is a rare example of a trans-acting regulator of poly(A) site choice. Analysing fpa mutants therefore provides an opportunity to reveal generic consequences of disrupting this process. We used direct RNA sequencing to quantify shifts in RNA 3′ formation in fpa mutants. Here we show that specific chimeric RNAs formed between the exons of otherwise separate genes are a striking consequence of loss of FPA function. We define intergenic read-through transcripts resulting from defective RNA 3′ end formation in fpa mutants and detail cryptic splicing and antisense transcription associated with these read-through RNAs. We identify alternative polyadenylation within introns that is sensitive to FPA and show FPA-dependent shifts in IBM1 poly(A) site selection that differ from those recently defined in mutants defective in intragenic heterochromatin and DNA methylation. Finally, we show that defective termination at specific loci in fpa mutants is shared with dicer-like 1 (dcl1) or dcl4 mutants, leading us to develop alternative explanations for some silencing roles of these proteins. We relate our findings to the impact that altered patterns of 3′ end formation can have on gene and genome organisation.

Resveratrol Protects Mouse Oocytes from Methylglyoxal-Induced Oxidative Damage

Methylglyoxal, a reactive dicarbonyl compound, is mainly formed from glycolysis. Methylglyoxal can lead to the dysfunction of mitochondria, the depletion of cellular anti-oxidation enzymes and the formation of advanced glycation ends. Previous studies showed that the accumulation of methylglyoxal and advanced glycation ends can impair the oocyte maturation and reduce the oocyte quality in aged and diabetic females. In this study, we showed that resveratrol, a kind of phytoalexin found in the skin of grapes, red wine and other botanical extracts, can alleviate the adverse effects caused by methylglyoxal, such as inhibition of oocyte maturation and disruption of spindle assembly. Besides, methylglyoxal-treated oocytes displayed more DNA double strands breaks and this can also be decreased by treatment of resveratrol. Further investigation of these processes revealed that methylglyoxal may affect the oocyte quality by resulting in excessive reactive oxygen species production, aberrant mitochondrial distribution and high level lipid peroxidation, and resveratrol can block these cytotoxic changes. Collectively, our results showed that resveratrol can protect the oocytes from methylglyoxal-induced cytotoxicity and this was mainly through the correction of the abnormity of cellular reactive oxygen species metabolism.

End Formation of a Round Tube into a Square Section with Reduced Forming Loads

Low formation loads are desirable in metal stamping industries as it reduces the press capacity of the machine and the tooling cost. In the previous study, the author had successfully developed a 2-stage end formation process of a round tube into a square section having small corner radii. However, the formation load in this process increased linearly with the punch stroke in the 1st stage due to the continuous expansion of the tube end by the conical die. Hence, buckling and cracks occurred at the circular section and the bottom end of the square section respectively when the punch stroke was excessive. In this study, the author proposes a circular die having a conical bottom replacing the conical die for the expansion of the tube end. Although the formation load increases when the tube end is expanded at the conical bottom, the amount of increase becomes small when the tube end reaches the circular section of the die due to its constant diameter. At the circular section, the tube end curls and wraps over the die when the punch stroke is increased. In the 2nd stage, the squaring process is performed with a conical bottom square punch and a taper square die for the two different expanded tubes i.e. the one formed with the conical die and the one formed with the conical bottom circular die. Both Finite Element Method (FEM) simulation and experiment were performed to evaluate these two processes. The distribution of plastic strains, forming loads and product appearances are investigated. With the circular die, the maximum forming loads are successfully reduced by 20% and 33% in the 1st and the 2nd stages respectively in the experiment when compared to the ones formed with the conical die. No buckling and cracks are observed for the tube formed with the circular die.

Regulation of HIV-1 gene expression

Gene expression is an immensely complicated process which requires fine tuning regulation involving multiple actors that have to work in a highly coordinated fashion. Indeed, each step of the transcription cycle (initiation, elongation and termination) is intimately coupled to a specif c step in pre-mRNA processing (5’ capping, splicing and 3’-end formation). Recent advances in the field highlighted the key role of the C-terminal domain (CTD) of the RNA polymerase II largest subunit in coordinating different steps of the transcription cycle and RNA processing. Transcription from the long terminal repeat (LTR) leads to RNAPII pausing and premature termination after synthesis of a short RNA, the transactivation response element (TAR). The HIV-1 transactivator protein, Tat, together with Cyclin T1 binds a bulge-loop within TAR leading to the recruitment of the super elongation complex (SEC) and allowing CDK9 to phosphorylate the RNAPII CTD and the negative transcription elongation factors NELF and the DRB-sensitivity-inducing factor DSIF/Spt4:Spt5 to overcome their negative action, licensing RNAPII for productive elongation. We will discuss how these recent advances might impact our view of how to design an efficient strategy leading to reactivation of transcriptionally silent HIV-1.

Polyadenylation site-specific differences in the activity of the neuronal βCstF-64 protein in PC-12 cells

Recent genome-wide analyses have implicated alternative polyadenylation — the process of regulated mRNA 3′ end formation — as a critical mechanism that influences multiple steps of mRNA metabolism in addition to increasing the protein-coding capacity of the genome. Although the functional consequences of alternative polyadenylation are well known, protein factors that regulate this process are poorly characterized. Previously, we described an evolutionarily conserved family of neuronal splice variants of the CstF-64 mRNA, βCstF-64, that we hypothesized to function in alternative polyadenylation in the nervous system. In the present study, we show that βCstF-64 mRNA and protein expression increase in response to nerve growth factor (NGF), concomitant with differentiation of adrenal PC-12 cells into a neuronal phenotype, suggesting a role for βCstF-64 in neuronal gene expression. Using PC-12 cells as model, we show that βCstF-64 is a bona fide polyadenylation protein, as evidenced by its association with the CstF complex, and by its ability to stimulate polyadenylation of luciferase reporter mRNA. Using luciferase assays, we show that βCstF-64 stimulates polyadenylation equivalently at the two weak poly(A) sites of the β-adducin mRNA. Notably, we demonstrate that the activity of βCstF-64 is less than CstF-64 on a strong polyadenylation signal, suggesting polyadenylation site-specific differences in the activity of the βCstF-64 protein. Our data address the polyadenylation functions of βCstF-64 for the first time, and provide initial insights into the mechanism of alternative poly(A) site selection in the nervous system.

A Transcriptome-wide Atlas of RNP Composition Reveals Diverse Classes of mRNAs and lncRNAs

SummaryEukaryotic genomes generate a heterogeneous ensemble of mRNAs and long noncoding RNAs (lncRNAs). LncRNAs and mRNAs are both transcribed by Pol II and acquire 5′ caps and poly(A) tails, but only mRNAs are translated into proteins. To address how these classes are distinguished, we identified the transcriptome-wide targets of 13 RNA processing, export, and turnover factors in budding yeast. Comparing the maturation pathways of mRNAs and lncRNAs revealed that transcript fate is largely determined during 3′ end formation. Most lncRNAs are targeted for nuclear RNA surveillance, but a subset with 3′ cleavage and polyadenylation features resembling the mRNA consensus can be exported to the cytoplasm. The Hrp1 and Nab2 proteins act at this decision point, with dual roles in mRNA cleavage/polyadenylation and lncRNA surveillance. Our data also reveal the dynamic and heterogeneous nature of mRNA maturation, and highlight a subset of “lncRNA-like” mRNAs regulated by the nuclear surveillance machinery.

Emerging roles for RNA polymerase II CTD in Arabidopsis

Post-translational modifications of the carboxy-terminal domain of the largest subunit of RNA polymerase II (RNAPII CTD) provide recognition marks to coordinate recruitment of numerous nuclear factors controlling transcription, cotranscriptional RNA processing, chromatin remodeling, and RNA export. Compared with the progress in yeast and mammals, deciphering the regulatory roles of position-specific combinatorial CTD modifications, the so-called CTD code, is still at an early stage in plants. In this review, we discuss some of the recent advances in understanding of the molecular mechanisms controlling the deposition and recognition of RNAPII CTD marks in plants during the transcriptional cycle and highlight some intriguing differences between regulatory components characterized in yeast, mammals, and plants.

R-loops and nicks initiate DNA breakage and genome instability in non-growing Escherichia coli.

Double-stranded DNA ends, often from replication, drive genomic instability, yet their origin in non-replicating cells is unknown. Here we show that transcriptional RNA/DNA hybrids (R-loops) generate DNA ends that underlie stress-induced mutation and amplification. Depleting RNA/DNA hybrids with overproduced RNase HI reduces both genomic changes, indicating RNA/DNA hybrids as intermediates in both. An Mfd requirement and inhibition by translation implicate transcriptional R-loops. R-loops promote instability by generating DNA ends, shown by their dispensability when ends are provided by I-SceI endonuclease. Both R-loops and single-stranded endonuclease TraI are required for end formation, visualized as foci of a fluorescent end-binding protein. The data suggest that R-loops prime replication forks that collapse at single-stranded nicks, producing ends that instigate genomic instability. The results illuminate how DNA ends form in non-replicating cells, identify R-loops as the earliest known mutation/amplification intermediate, and suggest that genomic instability during stress could be targeted to transcribed regions, accelerating adaptation.

The nuclear cap-binding complex interacts with the U4/U6·U5 tri-snRNP and promotes spliceosome assembly in mammalian cells

The nuclear cap-binding complex (CBC) binds to the 7-methyl guanosine cap present on every RNA polymerase II transcript. CBC has been implicated in many aspects of RNA biogenesis; in addition to roles in miRNA biogenesis, nonsense-mediated decay, 3'-end formation, and snRNA export from the nucleus, CBC promotes pre-mRNA splicing. An unresolved question is how CBC participates in splicing. To investigate CBC's role in splicing, we used mass spectrometry to identify proteins that copurify with mammalian CBC. Numerous components of spliceosomal snRNPs were specifically detected. Among these, three U4/U6·U5 snRNP proteins (hBrr2, hPrp4, and hPrp31) copurified with CBC in an RNA-independent fashion, suggesting that a significant fraction of CBC forms a complex with the U4/U6·U5 snRNP and that the activity of CBC might be associated with snRNP recruitment to pre-mRNA. To test this possibility, CBC was depleted from HeLa cells by RNAi. Chromatin immunoprecipitation and live-cell imaging assays revealed decreased cotranscriptional accumulation of U4/U6·U5 snRNPs on active transcription units, consistent with a requirement for CBC in cotranscriptional spliceosome assembly. Surprisingly, recruitment of U1 and U2 snRNPs was also affected, indicating that RNA-mediated interactions between CBC and snRNPs contribute to splicing. On the other hand, CBC depletion did not impair snRNP biogenesis, ruling out the possibility that decreased snRNP recruitment was due to changes in nuclear snRNP concentration. Taken together, the data support a model whereby CBC promotes pre-mRNA splicing through a network of interactions with and among spliceosomal snRNPs during cotranscriptional spliceosome assembly.

Non‐mRNA 3′ end formation: how the other half lives

The release of nascent RNA from transcribing RNA polymerase complexes is required for all further functions carried out by RNA molecules. The elements and processing machinery involved in 3' end formation therefore represent key determinants in the biogenesis and accumulation of cellular RNA. While these factors have been well-characterized for messenger RNA, recent work has elucidated analogous pathways for the 3' end formation of other important cellular RNA. Here, we discuss four specific cases of non-mRNA 3' end formation-metazoan small nuclear RNA, Saccharomyces cerevisiae small nuclear RNA, Schizosaccharomyces pombe telomerase RNA, and the mammalian MALAT1 large noncoding RNA-as models of alternative mechanisms to generate RNA 3' ends. Comparison of these disparate processing pathways reveals an emerging theme of evolutionary ingenuity. In some instances, evidence for the creation of a dedicated processing complex exists; while in others, components are utilized from the existing RNA processing machinery and modified to custom fit the unique needs of the RNA substrate. Regardless of the details of how non-mRNA 3' ends are formed, the lengths to which biological systems will go to release nascent transcripts from their DNA templates are fundamental for cell survival.

Splicing-coupled 3′ end formation requires a terminal splice acceptor site, but not intron excision

Splicing of human pre-mRNA is reciprocally coupled to 3′ end formation by terminal exon definition, which occurs co-transcriptionally. It is required for the final maturation of most human pre-mRNAs and is therefore important to understand. We have used several strategies to block splicing at specific stages in vivo and studied their effect on 3′ end formation. We demonstrate that a terminal splice acceptor site is essential to establish coupling with the poly(A) signal in a chromosomally integrated β-globin gene. This is in part to alleviate the suppression of 3′ end formation by U1 small nuclear RNA, which is known to bind pre-mRNA at the earliest stage of spliceosome assembly. Interestingly, blocks to splicing that are subsequent to terminal splice acceptor site function, but before catalysis, have little observable effect on 3′ end formation. These data suggest that early stages of spliceosome assembly are sufficient to functionally couple splicing and 3′ end formation, but that on-going intron removal is less critical.

Processing-Independent CRISPR RNAs Limit Natural Transformation in Neisseria meningitidis

CRISPR interference confers adaptive, sequence-based immunity against viruses and plasmids and is specified by CRISPR RNAs (crRNAs) that are transcribed and processed from spacer-repeat units. Pre-crRNA processing is essential for CRISPR interference in all systems studied thus far. Here, our studies of crRNA biogenesis and CRISPR interference in naturally competent Neisseria spp. reveal a unique crRNA maturation pathway in which crRNAs are transcribed from promoters that are embedded within each repeat, yielding crRNA 5' ends formed by transcription and not by processing. Although crRNA 3' end formation involves RNase III and trans-encoded tracrRNA, as in other type II CRISPR systems, this processing is dispensable for interference. The meningococcal pathway is the most streamlined CRISPR/Cas system characterized to date. Endogenous CRISPR spacers limit natural transformation, which is the primary source of genetic variation that contributes to immune evasion, antibiotic resistance, and virulence in the human pathogen N. meningitidis.

Abstract 4934: The role of Aurora A kinase-dependent phosphorylation of cortactin in invadopodia formation.

Abstract Aurora A kinase (AURKA) is a serine/threonine kinase known for its critical role in regulation of cell cycle progression. Overexpression and activation of AURKA is implicated in development of multiple types of human cancer. AURKA upregulation is considered as independent prognostic marker for breast cancer patients and associated with poor survival outcome. Recent work by our laboratory and others suggests a new role for AURKA in promotion of tumor cells migration and invasion through regulation of actin dynamics. However, the specific manner in which AURKA regulates actin-based machinery of migrating cancer cells is unknown. In our current work we show that phosphorylation of cortactin (CTTN) by AURKA directly regulates actin dynamics in breast cancer cells. CTTN is known to be regulated by oncogenic Src kinase. This phosphorylation is pivotal for formation of free barbed ends and further actin polymerization. Dynamic polymerization/depolimerization is required for invasion of cancer cells. Thus inhibition of Src-dependant phosphorylation of CTTN is critical for invadopodia maturation. In our current work we identify the phosphorylation site on CTTN targeted by AURKA and show that phosphorylation of CTTN by AURKA could potentially inhibit further phosphorylation of CTTN by Src leading to stabilization of invadopodia. Inhibition of AURKA activity or depletion of AURKA in cancer cells led to decrease in invasion and invadopodia maturation. In addition, we showed that adaptor protein NEDD9 which is known to bind both AURKA and Src also binds to CTTN and therefore potentially can mediate Src-CTTN-AURKA complex formation that regulates the sequential phosphorylation events necessary for invadopodia genesis. It is our expectation that AURKA-phosphorylated CTTN will inhibit Src-driven tyrosine-phosphorylation of CTTN, regulating a mechanistic switch between complementary actin polymerization and actin stabilization programs. Citation Format: Varvara K. Kozyreva, Laura Kelley, Sarah L. McLaughlin, Elena N. Pugacheva. The role of Aurora A kinase-dependent phosphorylation of cortactin in invadopodia formation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4934. doi:10.1158/1538-7445.AM2013-4934

A simple model to explain evolutionary trends of eukaryotic gene architecture and expression

Enormous phylogenetic variation exists in the number and sizes of introns in protein-coding genes. Although some consideration has been given to the underlying role of the population-genetic environment in defining such patterns, the influence of the intracellular environment remains virtually unexplored. Drawing from observations on interactions between co-transcriptional processes involved in splicing and mRNA 3'-end formation, a mechanistic model is proposed for splice-site recognition that challenges the commonly accepted intron- and exon-definition models. Under the suggested model, splicing factors that outcompete 3'-end processing factors for access to intronic binding sites concurrently favor the recruitment of 3'-end processing factors at the pre-mRNA tail. This hypothesis sheds new light on observations such as the intron-mediated enhancement of gene expression and the negative correlation between intron length and levels of gene expression.

End formation of a round tube into a square section having small corner radii

Expansion and reduction are the two common end forming processes for tubes. In the tube end expansion process using a square punch, it is difficult to obtain a small corner radii due to the stretching of the tube around the punch corners. The wall thickness around the corners is small when compared to the side wall. Hence, a tube having a poor square look is formed. In this study, a 2-stage end expansion of a round tube end into a square section having an improved square look i.e. small corner radii and increase in wall thickness around corners is developed. In the 1st stage, the tube end is flared into a cone shape using a 30° conical die by axial compression. In the 2nd stage, the conical end of the tube is drawn through a taper square die using a conical bottom square punch, and a near square section is formed. A 15% ironing ratio is applied during the drawing process to flatten the side wall of the square. Experimental and FEM simulation were performed to evaluate and to verify the forming process. Although the height of the square section increases when the punch stroke at the 1st stage is increased. However, this increase is limited by the buckling of the pipe at the circular section of the thick blank tube. Since the conical end is drawn into a square section having different radial lengths, the bottom of the square section is uneven. The uneven bottom end is trimmed off in the later process. A square section having a maximum height of 32mm after trimming is successfully obtained from the experiment for the punch stroke, S=44mm using an API 5L tube.

Characterization of snoRNA 3’‐end formation in Saccharomyces cerevisiae reveals a broad role for the Paf1 complex and locus‐specific roles for histone post‐translational modifications

One eukaryotic protein complex involved in regulating RNA polymerase II transcription is the Paf1 complex (Paf1C), which has well‐characterized roles in promoting histone modifications and transcription elongation. Beyond these roles, the human and yeast Paf1 complexes also interact with RNA 3’‐end processing components to affect RNA formation. Specifically, the S. cerevisiae Paf1C is known to promote the proper 3’‐end formation of at least two small nucleolar RNAs (snoRNAs). To determine how Paf1C‐dependent functions regulate formation of these non‐coding RNAs, we used high‐density tiling arrays to analyze transcripts in cells lacking PAF1 and uncover new snoRNA targets of Paf1. Increased levels of RNA polymerase II seen downstream of snoRNA genes in the absence of PAF1 suggest that these snoRNA 3’‐end defects reflect impaired transcription termination. Examination of Paf1‐regulated snoRNA genes revealed locus‐specific requirements for post‐translational histone modifications and for other transcriptional regulators. Cumulatively, our work reveals a broad requirement for Paf1C in snoRNA 3’‐end formation and suggests that Paf1C contributes to the fine‐tuning of transcriptional regulation at individual loci.

The Ameliorative Effects of L-2-Oxothiazolidine-4-Carboxylate on Acetaminophen-Induced Hepatotoxicity in Mice

The aim of the study was to investigate the ameliorative effects and the mechanism of action of l-2-oxothiazolidine-4-carboxylate (OTC) on acetaminophen (APAP)-induced hepatotoxicity in mice. Mice were randomly divided into six groups: normal control group, APAP only treated group, APAP + 25 mg/kg OTC, APAP + 50 mg/kg OTC, APAP + 100 mg/kg OTC, and APAP + 100 mg/kg N-acetylcysteine (NAC) as a reference control group. OTC treatment significantly reduced serum alanine aminotransferase and aspartate aminotransferase levels in a dose dependent manner. OTC treatment was markedly increased glutathione (GSH) production and glutathione peroxidase (GSH-px) activity in a dose dependent manner. The contents of malondialdehyde and 4-hydroxynonenal in liver tissues were significantly decreased by administration of OTC and the inhibitory effect of OTC was similar to that of NAC. Moreover, OTC treatment on APAP-induced hepatotoxicity significantly reduced the formation of nitrotyrosin and terminal deoxynucleotidyl transferase dUTP nick end labeling positive areas of liver tissues in a dose dependent manner. Furthermore, the activity of caspase-3 in liver tissues was reduced by administration of OTC in a dose dependent manner. The ameliorative effects of OTC on APAP-induced liver damage in mice was similar to that of NAC. These results suggest that OTC has ameliorative effects on APAP-induced hepatotoxicity in mice through anti-oxidative stress and anti-apoptotic processes.

The Thoc1 Encoded Ribonucleoprotein Is a Substrate for the NEDD4-1 E3 Ubiquitin Protein Ligase

Ribonucleoprotein (RNP) complexes form around nascent RNA during transcription to facilitate proper transcriptional elongation, RNA processing, and nuclear export. RNPs are highly heterogeneous, and different types of RNPs tend to package functionally related transcripts. These observations have inspired the hypothesis that RNP mediated mechanisms help specify coordinated gene expression. This hypothesis is supported by the observation that mutations in RNP components can cause defects in specific developmental pathways. How RNP biogenesis itself is regulated, however, is not well understood. The evolutionarily conserved THO RNP complex functions early during transcription to package nascent transcripts and facilitate subsequent RNP biogenesis. THO deficiency compromises transcriptional elongation as well as RNP mediated events like 3′ end formation and nuclear export for some transcripts. Using molecularly manipulated cells and in vitro reconstituted biochemical reactions, we demonstrate that the essential THO protein component encoded by the Thoc1 gene is poly-ubiquitinated by the NEDD4-1 E3 ubiquitin ligase. Poly-ubiquitinated pThoc1 is degraded by the proteasome. These results indicate THO activity is regulated by the ubiquitin-proteasome pathway, and that this regulation is evolutionarily conserved between yeast and mammals. Manipulation of NEDD4-1 levels has modest effects on Thoc1 protein levels under steady state conditions, but destabilization of Thoc1 protein upon treatment with a transcriptional elongation inhibitor is dependent on NEDD4-1. This suggests NEDD4-1 functions in conjunction with other post-translational mechanisms to regulate Thoc1 protein and THO activity.

Nuclear RNA surveillance: role of TRAMP in controlling exosome specificity

The advent of high-throughput sequencing technologies has revealed that pervasive transcription generates RNAs from nearly all regions of eukaryotic genomes. Normally, these transcripts undergo rapid degradation by a nuclear RNA surveillance system primarily featuring the RNA exosome. This multimeric protein complex plays a critical role in the efficient turnover and processing of a vast array of RNAs in the nucleus. Despite its initial discovery over a decade ago, important questions remain concerning the mechanisms that recruit and activate the nuclear exosome. Specificity and modulation of exosome activity requires additional protein cofactors, including the conserved TRAMP polyadenylation complex. Recent studies suggest that helicase and RNA-binding subunits of TRAMP direct RNA substrates for polyadenylation, which enhances their degradation by Dis3/Rrp44 and Rrp6, the two exosome-associated ribonucleases. These findings indicate that the exosome and TRAMP have evolved highly flexible functions that allow recognition of a wide range of RNA substrates. This flexibility provides the nuclear RNA surveillance system with the ability to regulate the levels of a broad range of coding and noncoding RNAs, which results in profound effects on gene expression, cellular development, gene silencing, and heterochromatin formation. This review summarizes recent findings on the nuclear RNA surveillance complexes, and speculates upon possible mechanisms for TRAMP-mediated substrate recognition and exosome activation.