End Formation Research Articles

A Subset of Drosophila Integrator Proteins Is Essential for Efficient U7 snRNA and Spliceosomal snRNA 3'-End Formation

A Subset of Drosophila Integrator Proteins Is Essential for Efficient U7 snRNA and Spliceosomal snRNA 3'-End Formation

An active role for splicing in 3′‐end formation

When intron-defined splicing was replaced by exon-defined splicing in the evolution of higher eukaryotes, the splicing apparatus had to rely on the cleavage/polyadenylation (CP) apparatus for help in defining the 3'-terminal exon. The 3'-terminal exon-definition complex that resulted consists of splicing factors on the upstream 3' splice site (ss) interacting with CP factors on the downstream poly(A) signal. A speculative model for assembly of this processing complex proposes several discrete steps. First, the splicing factor, U2AF65, interacts with the CP factor, CFI(m). Then, CFI(m) is displaced from U2AF65 by the poly(A) polymerase during a remodeling step. Finally, the U2 snRNP interacts with CPSF in a step resembling spliceosomal A-complex formation. The result is mutual enhancement of both splicing and CP for the exon. In contrast, when the poly(A) signal is preceded by a 5' rather than a 3' ss, competition replaces cooperation. Thus, a poly(A) site in an intron must compete with the upstream 5' ss for pairing with the 3' ss further upstream, across the presumptive exon. If the poly(A) site wins the competition, a terminal exon is defined. But if the 5' ss wins (by defining the upstream exon as internal, followed by pairing with a 3' ss across the downstream intron), then the poly(A) site is suppressed. The U1 snRNP obviously participates in this competition through its role in splice site pairing. However, the U1 snRNP can also bind elsewhere in the transcript, apart from splice sites, to regulate CP by direct interaction with the CP factors.

MRNA 3′ end processing and more—multiple functions of mammalian cleavage factor I‐68

The formation of defined 3(') ends is an important step in the biogenesis of mRNAs. In eukaryotic cells, all mRNA 3(') ends are generated by endonucleolytic cleavage of primary transcripts in reactions that are essentially posttranscriptional. Nevertheless, 3(') end formation is tightly connected to transcription in vivo, and a link with mRNA export to the cytoplasm has been postulated. Here, we briefly review the current knowledge about the two types of mRNA 3(') end processing reactions, cleavage/polyadenylation and histone RNA processing. We then focus on factors shared between these two reactions. In particular, we discuss evidence for new functions of the mammalian cleavage factor I subunit CF I(m) 68 in histone RNA 3(') processing and in the export of mature mRNAs from the nucleus to the cytoplasm.

Identification of transcription start sites of trans-spliced genes: Uncovering unusual operon arrangements

In Caenorhabditis elegans, the transcripts of many genes are trans-spliced to an SL1 spliced leader, a process that removes the RNA extending from the transcription start site to the trans-splice site, thereby making it difficult to determine the position of the promoter. Here we use RT-PCR to identify promoters of trans-spliced genes. Many genes in C. elegans are organized in operons where genes are closely clustered, typically separated by only ~100 nucleotides, and transcribed by an upstream promoter. The transcripts of downstream genes are trans-spliced to an SL2 spliced leader. The polycistronic precursor RNA is processed into individual transcripts by 3' end formation and trans-splicing. Although the SL2 spliced leader does not appear to be used for other gene arrangements, there is a relatively small number of genes whose transcripts are processed by SL2 but are not close to another gene in the same orientation. Although these genes do not appear to be members of classical C. elegans operons, we investigated whether these might represent unusual operons with long spacing or a different, nonoperon mechanism for specifying SL2 trans-splicing. We show transcription of the entire region between the SL2 trans-spliced gene and the next upstream gene, sometimes several kilobases distant, suggesting that these represent exceptional operons. We also report a second type of atypical "alternative" operon, in which 3' end formation and trans-splicing by SL2 occur within an intron. In this case, the processing sometimes results in a single transcript, and sometimes in two separate mRNAs.

3′ processing of eukaryotic precursor tRNAs

Biogenesis of eukaryotic tRNAs requires transcription by RNA polymerase III and subsequent processing. 5' processing of precursor tRNA occurs by a single mechanism, cleavage by RNase P, and usually occurs before 3' processing although some conditions allow observation of the 3'-first pathway. 3' processing is relatively complex and is the focus of this review. Precursor RNA 3'-end formation begins with pol III termination generating a variable length 3'-oligo(U) tract that represents an underappreciated and previously unreviewed determinant of processing. Evidence that the pol III-intrinsic 3'exonuclease activity mediated by Rpc11p affects 3'oligo(U) length is reviewed. In addition to multiple 3' nucleases, precursor tRNA(pre-tRNA) processing involves La and Lsm, distinct oligo(U)-binding proteins with proposed chaperone activities. 3' processing is performed by the endonuclease RNase Z or the exonuclease Rex1p (possibly others) along alternate pathways conditional on La. We review a Schizosaccharomyces pombe tRNA reporter system that has been used to distinguish two chaperone activities of La protein to its two conserved RNA binding motifs. Pre-tRNAs with structural impairments are degraded by a nuclear surveillance system that mediates polyadenylation by the TRAMP complex followed by 3'-digestion by the nuclear exosome which appears to compete with 3' processing. We also try to reconcile limited data on pre-tRNA processing and Lsm proteins which largely affect precursors but not mature tRNAs.A pathway is proposed in which 3' oligo(U) length is a primary determinant of La binding with subsequent steps distinguished by 3'-endo versus exo nucleases,chaperone activities, and nuclear surveillance.

Developments in RNA Splicing and Disease

Pre-mRNA processing, including 5'-end capping, splicing, editing, and polyadenylation, consists of a series of orchestrated and primarily cotranscriptional steps that ensure both the high fidelity and extreme diversity characteristic of eukaryotic gene expression. Alternative splicing and editing allow relatively small genomes to encode vast proteomic arrays while alternative 3'-end formation enables variations in mRNA localization, translation, and stability. Of course, this mechanistic complexity comes at a high price. Mutations in the myriad of RNA sequence elements that regulate mRNA biogenesis, as well as the trans-acting factors that act upon these sequences, underlie a number of human diseases. In this review, we focus on one of these key RNA processing steps, splicing, to highlight recent studies that describe both conventional and novel pathogenic mechanisms that underlie muscle and neurological diseases.

Sub1 Globally Regulates RNA Polymerase II C-Terminal Domain Phosphorylation

The transcriptional coactivator Sub1 has been implicated in several aspects of mRNA metabolism in yeast, such as activation of transcription, termination, and 3'-end formation. Here, we present evidence that Sub1 plays a significant role in controlling phosphorylation of the RNA polymerase II large subunit C-terminal domain (CTD). We show that SUB1 genetically interacts with the genes encoding all four known CTD kinases, SRB10, KIN28, BUR1, and CTK1, suggesting that Sub1 acts to influence CTD phosphorylation at more than one step of the transcription cycle. To address this directly, we first used in vitro kinase assays, and we show that, on the one hand, SUB1 deletion increased CTD phosphorylation by Kin28, Bur1, and Ctk1 but, on the other, it decreased CTD phosphorylation by Srb10. Second, chromatin immunoprecipitation assays revealed that SUB1 deletion decreased Srb10 chromatin association on the inducible GAL1 gene but increased Kin28 and Ctk1 chromatin association on actively transcribed genes. Taken together, our data point to multiple roles for Sub1 in the regulation of CTD phosphorylation throughout the transcription cycle.

Crosstalk between mRNA 3′ End Processing and Transcription Initiation

Transcription and mRNA maturation are interdependent events. Although stimulatory connections between these processes within the same round of transcription are well described, functional coupling between separate transcription cycles remains elusive. Comparing time-resolved transcription profiles of single-copy integrated β-globin gene variants, we demonstrate that a polyadenylation site mutation decreases transcription initiation of the same gene. Upon depletion of the 3' end processing and transcription termination factor PCF11, endogenous genes exhibit a similar phenotype. Readthrough RNA polymerase II (RNAPII) engaged on polyadenylation site-mutated transcription units sequester the transcription initiation/elongation factors TBP, TFIIB and CDK9, leading to their depletion at the promoter. Additionally, high levels of TBP and TFIIB appear inside the gene body, and Ser2-phosphorylated RNAPII accumulates at the promoter. Our data demonstrate that 3' end formation stimulates transcription initiation and suggest that coordinated recycling of factors from a gene terminator back to the promoter is essential for sustaining continued transcription.

Screening <i>Saccharomyces cerevisiae</i> for small-molecule inhibitors of mRNA 3’ end formation

mRNA 3’ end processing is an essential process shared by all eukaryotes and involves cleavage of the mRNAs and the addition of 70-90 adenosine residues. This modification is essential for mRNA biogenesis and gene expression. We developed a powerful screen to identify small molecule that inhibit mRNA 3’ end processing in yeast. Our growth-based assay employs a reporter plasmid with the CYC1 poly(A) site inserted upstream of the CUP1 coding sequence. In yeast carrying the reporter plasmid, defects in mRNA 3’ end processing are known to allow read-through into CUP1, enabling cells to grow in medium containing 0.6mM CuSO4. A high through-put screen of 70,000 compounds led to identification of 28 compounds. Different concentrations of each compound were then tested using the same assay to determine the dose response effect. Of those 28 compounds, 6 were confirmed to have a strong effect. b-galactosidase based assay was used to confirm the compounds that were effective in the growthbased assay and we found one compound (N,N-diethyl-N'-(4-methylbenzothioyl)thiourea) is effective in b-galactosidase assay and also strain defective in cleavage and polyadenylation is more sensitive for this compound more than wild type stain. Keywords: β-galactosidase assay; high-throughput screening; copper assay, mRNA 3’ end formation.

Nucleosome depletion at yeast terminators is not intrinsic and can occur by a transcriptional mechanism linked to 3'-end formation.

Genome-wide mapping of nucleosomes generated by micrococcal nuclease (MNase) suggests that yeast promoter and terminator regions are very depleted of nucleosomes, predominantly because their DNA sequences intrinsically disfavor nucleosome formation. However, MNase has strong DNA sequence specificity that favors cleavage at promoters and terminators and accounts for some of the correlation between occupancy patterns of nucleosomes assembled in vivo and in vitro. Using an improved method for measuring nucleosome occupancy in vivo that does not involve MNase, we confirm that promoter regions are strongly depleted of nucleosomes, but find that terminator regions are much less depleted than expected. Unlike at promoter regions, nucleosome occupancy at terminators is strongly correlated with the orientation of and distance to adjacent genes. In addition, nucleosome occupancy at terminators is strongly affected by growth conditions, indicating that it is not primarily determined by intrinsic histone-DNA interactions. Rapid removal of RNA polymerase II (pol II) causes increased nucleosome occupancy at terminators, strongly suggesting a transcription-based mechanism of nucleosome depletion. However, the distinct behavior of terminator regions and their corresponding coding regions suggests that nucleosome depletion at terminators is not simply associated with passage of pol II, but rather involves a distinct mechanism linked to 3'-end formation.

Alternative mRNA polyadenylation in eukaryotes: an effective regulator of gene expression

Alternative RNA processing mechanisms, including alternative splicing and alternative polyadenylation, are increasingly recognized as important regulators of gene expression. This article will focus on what has recently been described about alternative polyadenylation in development, differentiation, and disease in higher eukaryotes. We will also describe how the evolving global methodologies for examining the cellular transcriptome, both experimental and bioinformatic, are revealing new details about the complex nature of alternative 3(') end formation as well as interactions with other RNA-mediated and RNA processing mechanisms.

Coupled RNA polymerase II transcription and 3′ end formation with yeast whole-cell extracts

RNA polymerase II (RNAP II) transcription and pre-mRNA 3' end formation are linked through physical and functional interactions. We describe here a highly efficient yeast in vitro system that reproduces both transcription and 3' end formation in a single reaction. The system is based on simple whole-cell extracts that were supplemented with a hybrid Gal4-VP16 transcriptional activator and supercoiled plasmid DNA templates encoding G-less cassette reporters. We found that the coupling of transcription and processing in vitro enhanced pre-mRNA 3' end formation and reproduced requirements for poly(A) signals and polyadenylation factors. Unexpectedly, however, we show that in vitro transcripts lacked m⁷G-caps. Reconstitution experiments with CF IA factor assembled entirely from heterologous components suggested that the CTD interaction domain of the Pcf11 subunit was required for proper RNAP II termination but not 3' end formation. Moreover, we observed reduced termination activity associated with extracts prepared from cells carrying a mutation in the 5'-3' exonuclease Rat1 or following chemical inhibition of exonuclease activity. Thus, in vitro transcription coupled to pre-mRNA processing recapitulates hallmarks of poly(A)-dependent RNAP II termination. The in vitro transcription/processing system presented here should provide a useful tool to further define the role of factors involved in coupling.

Recent Insights in Pre-mRNA 3-End Processing Signals and Proteins in the Protozoan Parasite Entamoeba histolytica

The messenger RNA precursors (pre-mRNA) 3'-end processing occurs in a two-step co-transcriptional coupled reaction, denoted as cleavage and polyadenylation. Both processes depend on trans-acting factors interacting in a coordinated manner with cis-sequence motifs located at the 3' untranslated region of transcripts. In this paper, we reviewed mechanisms involved in pre-mRNA processing in eukaryotic organisms, including our own findings about sequences and proteins potentially involved in mRNA 3'-end formation in the protozoan parasite Entamoeba histolytica. Interestingly, protein sequence comparisons among E. histolytica, yeast, and human pre-mRNA processing machineries showed that amoeba pre-mRNA 3'-end processing machinery appears to be in an intermediate evolutionary position between mammals and yeast. In addition, the presence of non canonical poly(A) polymerases family recently identified in E. histolytica, adds more complexity to the mRNA 3'-end formation process in this ancient eukaryote.

Recognition of Polyadenosine RNA by the Zinc Finger Domain of Nuclear Poly(A) RNA-binding Protein 2 (Nab2) Is Required for Correct mRNA 3′-End Formation*

Proteins bound to the poly(A) tail of mRNA transcripts, called poly(A)-binding proteins (Pabs), play critical roles in regulating RNA stability, translation, and nuclear export. Like many mRNA-binding proteins that modulate post-transcriptional processing events, assigning specific functions to Pabs is challenging because these processing events are tightly coupled to one another. To investigate the role that a novel class of zinc finger-containing Pabs plays in these coupled processes, we defined the mode of polyadenosine RNA recognition for the conserved Saccharomyces cerevisiae Nab2 protein and assessed in vivo consequences caused by disruption of RNA binding. The polyadenosine RNA recognition domain of Nab2 consists of three tandem Cys-Cys-Cys-His (CCCH) zinc fingers. Cells expressing mutant Nab2 proteins with decreased binding to polyadenosine RNA show growth defects as well as defects in poly(A) tail length but do not accumulate poly(A) RNA in the nucleus. We also demonstrate genetic interactions between mutant nab2 alleles and mutant alleles of the mRNA 3′-end processing machinery. Together, these data provide strong evidence that Nab2 binding to RNA is critical for proper control of poly(A) tail length.

RNA Polymerase II C-Terminal Domain Phosphorylation Patterns in Caenorhabditis elegans Operons, Polycistronic Gene Clusters with Only One Promoter

The heptad repeat of the RNA polymerase II (RNAPII) C-terminal domain is phosphorylated at serine 5 near gene 5' ends and serine 2 near 3' ends in order to recruit pre-mRNA processing factors. Ser-5(P) is associated with gene 5' ends to recruit capping enzymes, whereas Ser-2(P) is associated with gene 3' ends to recruit cleavage and polyadenylation factors. In the gene clusters called operons in Caenorhabditis elegans, there is generally only a single promoter, but each gene in the operon forms a 3' end by the usual mechanism. Although downstream operon genes have 5' ends, they receive their caps by trans splicing rather than by capping enzymes. Thus, they are predicted to not need Ser-5 phosphorylation. Here we show by RNAPII chromatin immunoprecipitation (ChIP) that internal operon gene 5' ends do indeed lack Ser-5(P) peaks. In contrast, Ser-2(P) peaks occur at each mRNA 3' end, where the 3'-end formation machinery binds. These results provide additional support for the idea that the serine phosphorylation of the C-terminal domain (CTD) serves to bring RNA-processing enzymes to the transcription complex. Furthermore, these results provide a novel demonstration that genes in operons are cotranscribed from a single upstream promoter.

SnRNA 3′ end formation: the dawn of the Integrator complex

The ubiquitously expressed uridine-rich snRNAs (small nuclear RNAs) are essential for the removal of introns, proper expression of histone mRNA and biosynthesis of ribosomal RNA. Much is known about their assembly into snRNP (small nuclear ribonucleoprotein) particles and their ultimate function in the expression of other genes; however, in comparison, less is known about the biosynthesis of these critical non-coding RNAs. The sequence elements necessary for 3' end formation of snRNAs have been identified and, intriguingly, the processing of snRNAs is uniquely dependent on the snRNA promoter, indicating that co-transcriptional processing is important. However, the trans-acting RNA-processing factors that mediate snRNA processing remained elusive, hindering overall progress. Recently, the factors involved in this process were biochemically purified, and designated the Integrator complex. Since their initial discovery, Integrator proteins have been implicated not only in the production of snRNA, but also in other cellular processes that may be independent of snRNA biogenesis. In the present study, we discuss snRNA biosynthesis and the roles of Integrator proteins. We compare models of 3' end formation for different classes of RNA polymerase II transcripts and formulate/propose a model of Integrator function in snRNA biogenesis.

Subsidence Control of Construction on Soft Soils with “Akar Foundation”

Good quality soils are always preferred in development projects, where the bearing capacity of the grounds is sufficiently high and the resulting subsidence is non-excessive. However these sites may not be as readily available with increased population and land use, making it inevitable to construct on less favourable soils, like soft organic materials. To ensure long term stability of structures erected on such soft soils, elaborate and extensive foundation systems are generally required. Alternatively, pre-treatment with ground improvement techniques is necessary. Both these methods tend to incur high costs and labour as well as require prolonged construction period, while making a rather negative impact on the environment in terms of raw material sourcing, heavy machinery mobilization and exposure to the risk of groundwater contamination. This paper describes an exploratory study on a simple shallow foundation system for construction on soft soils, which can be a potentially cheaper and more sustainable approach. The foundation system, termed the “Akar Foundation”, literally translates as “Root Foundation”, is essentially a lightweight platform supported by a group of hollow stumps (i.e. PVC pipes). The ‘root’ base served dual functions: 1. to collectively assert a stronger grip of the soft soils, hence giving higher bearing capacity; 2. to spread the imposed structural load evenly into the subsoil, thus avoiding excessive and non-uniform settlements. The effects of the end condition of the pipes (i.e. open or close) as well as the spacing between the pipes on the foundation settlement mechanism were examined, with static load tests conducted using a lab-scale simulation chamber. The findings showed that the effectiveness of the “Akar Foundation” depends on the compatibility of the pipe spacing and individual pipe lengths, highlighting the inter-relationship between the mobilisation of end resistance, skin friction and formation of soil plug in the open-end system. In a promising light, the reduced subsidence suggest the potential of the “Akar Foundation” as an economical yet effective foundation system in economically challenged soft soil areas.

High glucose activates HIF-1-mediated signal transduction in glomerular mesangial cells through a carbohydrate response element binding protein

High glucose evokes a variety of signals in mesangial cells that alter cellular functions responsible for the development of diabetic glomerulopathy. The hypoxia-inducible factor-1alpha (HIF-1alpha) regulates cellular homeostasis under hypoxic conditions, but it also has pleiotropic effects in response to cellular stresses at normoxia. Here we determined whether HIF-1alpha has a role in the regulation of mesangial cells in hyperglycemia. In the streptozotocin-induced diabetic mouse model, glomerular mesangial cells had a significant increase in HIF-1alpha expression in the nucleus. In cultured mesangial cells, high glucose enhanced the expression of HIF-1alpha and its target genes known to be involved in the development of diabetic glomerulopathy. A glucose-responsive carbohydrate response element binding protein (ChREBP) was found to have a critical role in the transcriptional upregulation of HIF-1alpha and downstream gene expression in mesangial cells exposed to high glucose. Knockdown of HIF-1alpha or ChREBP in mesangial cells abrogated the high glucose-mediated perturbation of gene expression. Our results show that ChREBP and HIF-1alpha mediate gene regulation in mesangial cells. Further studies will be needed to find out whether these findings are relevant to the development of the diabetic nephropathy.

MEASUREMENT OF DISSOLVED INORGANIC NUTRIENT IN EUPHOTIC ZONE THE BANTEN BAY

At a few this last years, measurement concentration of dissolved inorganic nutrient is has rapidlyed grow by using various methods. But method anything applied must based on at formation of indicator end of measurement amonia, nitrite, nitrate, orthophosphate and silicate. At measurement of amonia its indicator is formation of blue indophenol, nitrite formation of pink azo, nitrate formation of yellow colored solution, orthophosphate formation of blue molybdenum, and silicate based on formation of yellows silicomolibdate. The intensity of color that is highly dependent on the concentration of each element. Measurement of amonia blue indophenol intensity perfected concentration in the range 0.206-0.396 mg/L. Measurement nitrite the formation of a pink azo easy imperfect because its low concentration. Intensity measurements nitrate solution yellow perfect concentration in the range 0.128-0.989 mg/L. Measurement of imperfect blue molybdenum intensity orthophosphate because its low concentration. While measuring the formation silicate of yellow silicomolibdate perfect concentration on the range 10.573-26.470 mg/L. As a whole from result of measurement is obtained chemical composition of dissolved inorganic nutrient in euphotic zone the Banten bay is more predominated by silicate 97.27%, nitrate 1.84%, amonia 0.49%, orthophosphate 0.20%, and nitrite 0.18%. Keywords: blue indophenol, pink azo, yellow solution, blue molybdenum, yellow silicomolibdate

Cotranscriptional Rna Checkpoints

Transcription of protein-coding genes by RNA polymerase II is a repetitive, cyclic process that enables synthesis of multiple RNA molecules from the same template. The transcription cycle consists of three main stages, initiation, elongation and termination. Each of these phases is intimately coupled to a specific step in pre-mRNA processing; 5´ capping, splicing and 3´-end formation, respectively. In this article, we discuss the recent concept that cotranscriptional checkpoints operate during mRNA biogenesis to ensure that nonfunctional mRNAs with potentially deleterious effects for the cell are not produced or exported to the cytoplasm for translation.