scholarly journals Precise gene models using long-read sequencing reveal a unique poly(A) signal in Giardia lamblia

2021 ◽  
Author(s):  
Danielle Y Bilodeau ◽  
Ryan M Sheridan ◽  
Balu Balan ◽  
Aaron R Jex ◽  
Olivia S Rissland
Keyword(s):  
Gene Expression ◽  
Protein Complex ◽  
Giardia Lamblia ◽  
Alternative Polyadenylation ◽  
Mrna Processing ◽  
Cleavage Sites ◽  
Nascent Transcript ◽  
Long Read ◽  
Show Evidence ◽  
Cleavage And Polyadenylation

During pre-mRNA processing, the poly(A) signal is recognized by a protein complex that ensures precise cleavage and polyadenylation of the nascent transcript. The location of this cleavage event establishes the length and sequence of the 3' UTR of an mRNA, thus determining much of its post-transcriptional fate. Here, using long-read sequencing, we characterize the polyadenylation signal and related sequences surrounding Giardia lamblia cleavage sites for over 2600 genes. We find that G. lamblia uses a AGURAA poly(A) signal, which differs from the mammalian AAUAAA. We also describe how G. lamblia lacks common auxiliary elements found in other eukaryotes, along with the proteins that recognize them. Further, we identify 133 genes that show evidence of alternative polyadenylation. These results suggest that despite pared down cleavage and polyadenylation machinery, 3' end formation still appears to be an important regulatory step for gene expression in G. lamblia.

scholarly journals The Detection and Bioinformatic Analysis of Alternative 3′ UTR Isoforms as Potential Cancer Biomarkers

2021 ◽  
Vol 22 (10) ◽  
pp. 5322
Author(s):  
Nitika Kandhari ◽  
Calvin A. Kraupner-Taylor ◽  
Paul F. Harrison ◽  
David R. Powell ◽  
Traude H. Beilharz
Keyword(s):  
Gene Expression ◽  
Cell Transformation ◽  
Alternative Polyadenylation ◽  
Cancer Biomarkers ◽  
Bioinformatic Analysis ◽  
Alternative Transcript ◽  
Clinical Parameters ◽  
Transcript Cleavage ◽  
Cleavage And Polyadenylation ◽  
Potential Cancer

Alternative transcript cleavage and polyadenylation is linked to cancer cell transformation, proliferation and outcome. This has led researchers to develop methods to detect and bioinformatically analyse alternative polyadenylation as potential cancer biomarkers. If incorporated into standard prognostic measures such as gene expression and clinical parameters, these could advance cancer prognostic testing and possibly guide therapy. In this review, we focus on the existing methodologies, both experimental and computational, that have been applied to support the use of alternative polyadenylation as cancer biomarkers.

scholarly journals SRm160 Splicing Coactivator Promotes Transcript 3′-End Cleavage

2002 ◽  
Vol 22 (1) ◽  
pp. 148-160 ◽  
Author(s):  
Susan McCracken ◽  
Mark Lambermon ◽  
Benjamin J. Blencowe
Keyword(s):  
Gene Expression ◽  
Matrix Protein ◽  
Mrna Processing ◽  
Nuclear Matrix Protein ◽  
Mrna Transcripts ◽  
Cleavage And Polyadenylation ◽  
Specificity Factor ◽  
Cytoplasmic Accumulation

ABSTRACT Individual steps in the processing of pre-mRNA, including 5′-end cap formation, splicing, and 3′-end processing (cleavage and polyadenylation) are highly integrated and can influence one another. In addition, prior splicing can influence downstream steps in gene expression, including export of mRNA from the nucleus. However, the factors and mechanisms coordinating these steps in the maturation of pre-mRNA transcripts are not well understood. In the present study we demonstrate that SRm160 (for serine/arginine repeat-related nuclear matrix protein of 160 kDa), a coactivator of constitutive and exon enhancer-dependent splicing, participates in 3′-end formation. Increased levels of SRm160 promoted the 3′-end cleavage of transcripts both in vivo and in vitro. Remarkably, at high levels in vivo SRm160 activated the 3′-end cleavage and cytoplasmic accumulation of unspliced pre-mRNAs, thereby uncoupling the requirement for splicing to promote the 3′-end formation and nuclear release of these transcripts. Consistent with a role in 3′-end formation coupled to splicing, SRm160 was found to associate specifically with the cleavage polyadenylation specificity factor and to stimulate the 3′-end cleavage of splicing-active pre-mRNAs more efficiently than that of splicing-inactive pre-mRNAs in vitro. The results provide evidence for a role for SRm160 in mRNA 3′-end formation and suggest that the level of this splicing coactivator is important for the proper coordination of pre-mRNA processing events.

An Intronic Suppressor Element Regulates RUNX1 Alternative Polyadenylation

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3578-3578
Author(s):  
Amanda Scholl ◽  
Alexander Muselman ◽  
Dong-Er Zhang
Keyword(s):  
Gene Expression ◽  
Binding Protein ◽  
Alternative Polyadenylation ◽  
Regulatory Elements ◽  
Therapeutic Benefit ◽  
Mrna Processing ◽  
Alternative Exon ◽  
Constitutive Exon ◽  
Hematopoietic Stem ◽  
Polypyrimidine Tract Binding Protein

Abstract Polyadenylation is a post-transcriptional modification where the 3' end of an mRNA is cleaved and 250-300 adenines are added. It is predicted that 70-75% of human genes have more than one polyadenylation sequence (PAS) and are subject to alternative polyadenylation (APA). APA events affect the coding sequence of a gene when a proximal PAS is located within an intron, constitutive exon, or alternative exon. Gene expression is also affected if there are multiple PAS within the distal 3' untranslated region (UTR); proximal PAS usage shortens the 3'UTR, which can remove cis-regulatory regions such as miRNA and RNA-binding protein (RBP) sites. Furthermore, global changes in APA are linked to cellular state-proximal PAS usage is associated with immature developmental phases, cell proliferation, and cancerous phenotypes. Consequently, APA is a pertinent post-transcriptional modification that regulates gene expression and isoform generation across developmental stages and tissue types. Despite its significance, there are few APA studies in the hematology field, and those that exist have focused on global shifts in PAS usage. In this study, we uniquely focus on the APA mechanism of a single gene, RUNX1, and how this event can alter hematopoietic stem cell (HSC) homeostasis and hematopoiesis. There are three main isoforms of RUNX1 that differ in promoter and/or PAS usage. RUNX1b/c use different promoters, but have identical C-terminal regions. RUNX1a utilizes the same promoter as RUNX1b, but differs from both RUNX1b/c due to usage of a proximal PAS located in alternative exon 7a. RUNX1b/c are robustly expressed in most progenitor populations and differentiated blood cell lineages, whereas RUNX1a is restricted to human CD34+ HSCs. Functionally, RUNX1b/c promote HSC differentiation and lineage commitment, whereas RUNX1a expands HSCs and their engraftment potential, a property with therapeutic advantages but leukemic potential. Due to the difference in expression pattern and distinct functionality of RUNX1a compared to RUNX1b/c, it is relevant to study the APA event that dictates isoform generation. Elucidating this mechanism could provide valuable insight into the transient control of the HSC population for therapeutic benefit and illuminate new leukemogenic pathways. To study RUNX1 APA, we cloned alternative terminal exon 7a (RUNX1a) and constitutive exon 7b (RUNX1b/c) in between the two exons of a split GFP minigene reporter, along with 500 bp of their upstream and downstream flanking introns. We hypothesized that exon 7a would be skipped during processing of the minigene construct because the proximal PAS is rarely used in vivo. Conversely, exon 7b, the penultimate exon in RUNX1b/c, would be spliced in between the GFP exons, disrupting the GFP protein. These constructs were tested in KG-1a and U937 cells. Flow cytometry for GFP fluorescence supported our hypothesis as the exon 7a minigene produced a robust GFP signal and the exon 7b minigene produced no GFP signal. We confirmed that the GFP changes were due to the hypothesized mRNA processing events by performing RT-PCR using primers specific to the two GFP exons. These data show that important cis-regulatory elements that determine RUNX1 APA are located within exon 7a, 7b, and the cloned intronic regions. Next, we altered these minigenes by strategically making chimeric constructs that consist of either exon 7a or 7b with all combinations of upstream/downstream flanking introns. We discovered that replacing the intron upstream of exon 7a confers 2-5 fold greater splicing and polyadenylation of exon 7a, indicative of RUNX1a isoform generation. Therefore, a suppressor cis-element is located in this upstream intronic region. However, placing this intron upstream of exon 7b is not sufficient to reduce its inclusion between the GFP exons. Instead, both the upstream and downstream intronic regions flanking exon 7a are required. This suggests an RNA-looping mechanism that prevents splicing and usage of the exon 7a proximal PAS. Cleavage factor (CFIm) and Polypyrimidine-tract binding protein 1 (PTBP1) are RBPs involved in splicing and polyadenylation that alter mRNA processing by RNA-looping. We aim to narrow down the suppressor region upstream of exon 7a to identify a consensus sequence and the respective RBP that diminishes RUNX1 proximal PAS usage. This knowledge can be leveraged to enhance RUNX1a production and expand HSCs for therapeutic benefit. Disclosures No relevant conflicts of interest to declare.

scholarly journals Definition of essential sequences and functional equivalence of elements downstream of the adenovirus E2A and the early simian virus 40 polyadenylation sites.

1985 ◽  
Vol 5 (11) ◽  
pp. 2975-2983 ◽  
Author(s):  
R P Hart ◽  
M A McDevitt ◽  
H Ali ◽  
J R Nevins
Keyword(s):  
Essential Element ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Cleavage Sites ◽  
Multiple Sequence ◽  
Polyadenylic Acid ◽  
Sequence Elements ◽  
Cleavage And Polyadenylation ◽  
Definition Of ◽  
A Site

In addition to the highly conserved AATAAA sequence, there is a requirement for specific sequences downstream of polyadenylic acid [poly(A)] cleavage sites to generate correct mRNA 3' termini. Previous experiments demonstrated that 35 nucleotides downstream of the E2A poly(A) site were sufficient but 20 nucleotides were not. The construction and assay of bidirectional deletion mutants in the adenovirus E2A poly(A) site indicates that there may be redundant multiple sequence elements that affect poly(A) site usage. Sequences between the poly(A) site and 31 nucleotides downstream were not essential for efficient cleavage. Further deletion downstream (3' to +31) abolished efficient cleavage in certain constructions but not all. Between +20 and +38 the sequence T(A/G)TTTTT was duplicated. Function was retained when one copy of the sequence was present, suggesting that this sequence represents an essential element. There may also be additional sequences distal to +43 that can function. To establish common features of poly(A) sites, we also analyzed the early simian virus 40 (SV40) poly(A) site for essential sequences. An SV40 poly(A) site deletion that retained 18 nucleotides downstream of the cleavage site was fully functional while one that retained 5 nucleotides downstream was not, thus defining sequences required for cleavage. Comparison of the SV40 sequences with those from E2A did not reveal significant homologies. Nevertheless, normal cleavage and polyadenylation could be restored at the early SV40 poly(A) site by the addition of downstream sequences from the adenovirus E2A poly(A) site to the SV40 +5 mutant. The same sequences that were required in the E2A site for efficient cleavage also restored activity to the SV40 poly(A) site.

MAAPER: model-based analysis of alternative polyadenylation using 3' end-linked reads

2021 ◽  
Author(s):  
Wei Vivian Li ◽  
Dinghai Zheng ◽  
Ruijia Wang ◽  
Bin Tian
Keyword(s):  
Single Cell ◽  
Robust Statistics ◽  
Single Cell Analysis ◽  
Alternative Polyadenylation ◽  
Computational Method ◽  
Model Based ◽  
Eukaryotic Genes ◽  
Counting Strategy ◽  
Cleavage And Polyadenylation ◽  
Model Based Analysis

Most eukaryotic genes harbor multiple cleavage and polyadenylation sites (PASs), leading to expression of alternative polyadenylation (APA) isoforms. APA regulation has been implicated in a diverse array of physiological and pathological conditions. While RNA sequencing tools that generate reads containing the PAS, named onSite reads, have been instrumental in identifying PASs, they have not been widely used. By contrast, a growing number of methods generate reads that are close to the PAS, named nearSite reads, including the 3' end counting strategy commonly used in single cell analysis. How these nearSite reads can be used for APA analysis, however, is poorly studied. Here, we present a computational method, named model-based analysis of alternative polyadenylation using 3' end-linked reads (MAAPER), to examine APA using nearSite reads. MAAPER uses a probabilistic model to predict PASs for nearSite reads with high accuracy and sensitivity, and examines different types of APA events, including those in 3'UTRs and introns, with robust statistics. We show MAAPER's accuracy with data from both bulk and single cell RNA samples and its applicability in unpaired or paired experimental designs. Our result also highlights the importance of using well annotated PASs for nearSite read analysis.

Alternative translation and alternative RNA processing mechanism in parvovirus RNA processing

2014 ◽  
Author(s):  
◽  
Olufemi Fasina
Keyword(s):  
Gene Expression ◽  
Rna Processing ◽  
Viral Genome ◽  
Transcription Initiation ◽  
Alternative Polyadenylation ◽  
Open Reading Frames ◽  
Genome Replication ◽  
University Of Missouri ◽  
Diverse Range ◽  
Viral Genome Replication

[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT AUTHOR'S REQUEST.] Viruses as obligate intracellular metabolic parasite require the capacity to orchestrate and modulate the host environment either in the nucleus or cytoplasm for their efficient reproductive life cycle. This warrants the use of diverse range of proteins expressed from the viral genome with the ability of regulating viral genome replication, transcription and translation, in addition antagonizing host factors inhibitory to the virus. Therefore, in order to achieve these goals, viruses utilizes gene expression strategies to expand their coding capacity. Gene expression mechanism such as transcription initiation, capping, splicing and 3�-end processing afford viruses the opportunities to utilize the eukaryotic metabolic machineries for generating proteome diversity. Parvoviruses and other DNA viruses effectively capitalize on their use of nuclear eukaryotic metabolic machineries to co-opt host cell factors for optimal replication and gene expression. Parvoviruses with small genome size and overlapping open reading frames utilize alternative transcription initiation, alternative splicing and alternative polyadenylation to co-ordinate the expression of its non-structural and structural proteins. In this work, we have characterized how two parvoviruses; Dependovirus AAV5 and Bocavirus Minute virus of canine (MVC) utilize alternative gene expression mechanisms and strategies to optimize expression of viral proteins from their genome.

Identification of CCNB2 expression in triple-negative breast cancer based on bioinformatics results

2021 ◽  
Author(s):  
jintao cao ◽  
SHUAI SUN ◽  
RAN LI ◽  
RUI MIN ◽  
XINGYU FAN ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Gene Expression ◽  
Triple Negative Breast Cancer ◽  
Protein Complex ◽  
Triple Negative ◽  
Expression Profiles ◽  
Pathway Enrichment Analysis ◽  
Analysis Tool ◽  
The Core ◽  
Core Genes

Abstract Background The current epidemiology shows that the incidence of breast cancer is increasing year by year and tends to be younger. Triple-negative breast cancer is the most malignant of breast cancer subtypes. The application of bioinformatics in tumor research is becoming more and more extensive. This study provided research ideas and basis for exploring the potential targets of gene therapy for triple-negative breast cancer (TNBC). Methods We analyzed three gene expression profiles (GSE64790、GSE62931、GSE38959) selected from the Gene Expression Omnibus (GEO) database. The GEO2R online analysis tool was used to screen for differentially expressed genes (DEGs) between TNBC and normal tissues. Gene Ontology (GO) function and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were applied to identify the pathways and functional annotation of DEGs. Protein–protein interaction network of these DEGs were visualized by the Metascape gene-list analysis tool so that we could find the protein complex containing the core genes. Subsequently, we investigated the transcriptional data of the core genes in patients with breast cancer from the Oncomine database. Moreover, the online Kaplan–Meier plotter survival analysis tool was used to evaluate the prognostic value of core genes expression in TNBC patients. Finally, immunohistochemistry (IHC) was used to evaluated the expression level and subcellular localization of CCNB2 on TNBC tissues. Results A total of 66 DEGs were identified, including 33 up-regulated genes and 33 down-regulated genes. Among them, a potential protein complex containing five core genes was screened out. The high expression of these core genes was correlated to the poor prognosis of patients suffering breast cancer, especially the overexpression of CCNB2. CCNB2 protein positively expressed in the cytoplasm, and its expression in triple-negative breast cancer tissues was significantly higher than that in adjacent tissues. Conclusions CCNB2 may play a crucial role in the development of TNBC and has the potential as a prognostic biomarker of TNBC.

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