Abstract 17448: Comprehensive Characterization of Alternative Polyadenylation in Human Right Ventricle Failure

Background: Alternative cleavage and polyadenylation (APA) an emerging post-transcriptional mechanism for gene regulation that generates distinct isoforms of mRNA with different 3’UTR lengths. APA plays an important role in different biological processes and dysregulation of APA leading to many human diseases. However, the functional consequences of APA events in the right ventricle (RV) failure in humans remain unexplored. Objective: To identify whether the 3′UTR length is modulated by APA in the RV failure in humans compared to healthy RV. Methods and Results: We used Poly A tail RNA sequencing and a novel algorithm called DaPars to measure the global patterns of APA in healthy and failing human RV specimens. We revealed 3'UTR shortening and lengthening of many genes in failing RV compared to healthy RV specimens. The lengthened 3’UTR genes were enriched for functional groups such as RNA processing and splicing. Whereas the shortened 3’UTR genes were enriched for nucleic acid metabolic process and chromatin organization. In addition, differential APA events in RV failure regulates many pathways important for the progression of the RV failure. Finally, the regulator proteins of APA including cleavage and polyadenylation specificity factor (CPSF) 6 and 7, cleavage factor Im (CFIm) 25 and 59 have been regulated in RV failure compared to healthy RV specimens. Conclusions: Our results highlight important roles of APA in the progression of RV failure in humans, including RNA processing, splicing, and specific gene expression. Demonstrating that APA mediated 3’UTR length regulation provides the additional layer of gene expressions in RV failure.

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Abstract P507: Comprehensive Characterization Of Poly(a)-clickseq Data Sets Reveal Novel Polyadenylation Signals In Human Right Ventricle Failure

Circulation Research ◽

10.1161/res.129.suppl_1.p507 ◽

2021 ◽

Vol 129 (Suppl_1) ◽

Author(s):

Rajarajan A Amirthalingam Thandavarayan ◽

Hari Krishna Yalamanchili ◽

Rajasekaran Mahalingam ◽

Scott D Collum ◽

Keith Youker ◽

...

Keyword(s):

Right Ventricle ◽

Alternative Polyadenylation ◽

Specific Gene ◽

Significant Shift ◽

Data Sets ◽

Human Right ◽

Tissue Samples ◽

Human Genes ◽

Healthy Donors ◽

The Right

Background: Many human genes hold more than 1 poly(A) site, and polyadenylation gives rise to transcripts with different 3′ untranslated regions (3’UTR) lengths, and this process known as alternative polyadenylation (APA). It plays a significant role in many cellular processes and dysregulation of APA leads to many human diseases. However, the functional consequences of APA events in the right ventricle (RV) failure in humans remain unexplored. Objective: To understand whether a global APA event is presented in the progression of RV failure by using the novel algorithm PolyA miner in the RV of healthy donors and patients with RV failure. Methods and Results: RV tissue samples were obtained from failing human hearts at the time of cardiac transplantation at the Houston Methodist DeBakey Heart & Vascular Center, Houston Methodist Hospital. The RV tissues were immediately frozen in liquid nitrogen and stored at –80°C until use. Normal tissue samples were obtained from donor hearts that were not used for transplantation and were collected and stored in the same manner. We characterized the APA profiles of RV failing patients and compared them to healthy RV specimens by using Poly(A)-ClickSeq (PAC-seq) RNA sequencing and the PolyA-miner algorithm. We determined shortening versus lengthening of 3′UTRs based on the PolyA index, a metric unit that determines the length of 3′UTR. Based on these scores, we identified 435 transcripts with a significant shift in cleavage site usage. Further, we discovered 3'UTR shortening and lengthening of many genes in failing RV compared to healthy RV specimens. By examining polyadenylation events in these hearts, we identified disease-specific APA signatures in many genes. In addition, differential APA events in RV failure regulate many pathways important for the progression of the RV failure. Conclusions: Our study highlights the important roles for APA in human RV failure, including reforming multiple pathways and regulating specific gene expression, representing the complex interplay between APA and other biological processes in the RV failure.

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Abstract P501: Comprehensive Analysis Of Alternative Polyadenylation In Human Left Ventricle Failure By Using The Novel Algorithm: Polya-miner

Circulation Research ◽

10.1161/res.129.suppl_1.p501 ◽

2021 ◽

Vol 129 (Suppl_1) ◽

Author(s):

Rahul Neupane ◽

Hari Krishna Yalamanchili ◽

Rajasekaran Mahalingam ◽

Scott D Collum ◽

Keith Youker ◽

...

Keyword(s):

Left Ventricle ◽

Alternative Polyadenylation ◽

Significant Shift ◽

Gene Expressions ◽

Additional Layer ◽

Functional Consequences ◽

Cleavage And Polyadenylation ◽

Length Regulation ◽

Specificity Factor ◽

Human Left Ventricle

Background: Alternative polyadenylation (APA) is an emerging post-transcriptional mechanism for gene regulation that generates distinct isoforms of mRNA with different 3′ untranslated regions (3’UTR) lengths. APA plays an important role in different biological processes and dysregulation of APA leads to many human diseases. However, the functional consequences of APA events in the left ventricle (LV) failure in humans remain unexplored. Objective: To identify whether the 3′UTR length is modulated by APA in the LV failure in humans compared to healthy LV. Methods and Results: We used Poly(A)-ClickSeq RNA sequencing and PolyA-miner algorithm to measure the global patterns of APA in healthy and failing human LV specimens. We determined shortening versus lengthening of 3′UTRs based on the PolyA index, a metric unit that determines the length of 3′UTR. Based on these scores, we identified 129 genes with a significant shift of cleavage site usage in failing LV compared to healthy LV specimens. By examining polyadenylation events in these hearts, we identified disease-specific APA signatures in many genes. In addition, differential APA events in LV failure regulate many pathways important for the progression of LV failure. Finally, the regulator proteins of APA including cleavage and polyadenylation specificity factor (CPSF) 6 and 7, cleavage factor Im (CFIm) 25 and 59 have been regulated in LV failure compared to healthy LV specimens. Conclusions: Our results provide genome-wide, polyadenylation maps of the human heart and show that APA of mRNA is dynamic in the progression of LV failure in humans. Demonstrating that APA mediated 3’UTR length regulation provides the additional layer of gene expressions in LV failure.

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Transmural heterogeneity of action potentials andI to1 in myocytes isolated from the human right ventricle

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.1998.275.2.h369 ◽

1998 ◽

Vol 275 (2) ◽

pp. H369-H377 ◽

Cited By ~ 64

Author(s):

Gui-Rong Li ◽

Jianlin Feng ◽

Lixia Yue ◽

Michel Carrier

Keyword(s):

Right Ventricle ◽

Cardiac Electrophysiology ◽

Cell Types ◽

M Cells ◽

Limited Information ◽

Human Right ◽

Dependent Inactivation ◽

Significant Difference ◽

The Right ◽

Transmural Heterogeneity

Limited information is available about transmural heterogeneity in cardiac electrophysiology in man. The present study was designed to evaluate heterogeneity of cardiac action potential (AP), transient outward K+ current ( I to1) and inwardly rectifying K+ current ( I K1) in human right ventricle. AP and membrane currents were recorded using whole cell current- and voltage-clamp techniques in myocytes isolated from subepicardial, midmyocardial, and subendocardial layers of the right ventricle of explanted failing human hearts. AP morphology differed among the regional cell types. AP duration (APD) at 0.5–2 Hz was longer in midmyocardial cells (M cells) than in subepicardial and subendocardial cells. At room temperature, observed I to1, on step to +60 mV, was significantly greater in subepicardial (6.9 ± 0.8 pA/pF) and M cells (6.0 ± 1.1 pA/pF) than in subendocardial cells (2.2 ± 0.7 pA/pF, P < 0.01). Slower recovery of I to1 was observed in subendocardial cells. The half-inactivation voltage of I to1 was more negative in subendocardial cells than in M and subepicardial cells. At 36°C, the density of I to1 increased, the time-dependent inactivation and reactivation accelerated, and the frequency-dependent reduction attenuated in all regional cell types. No significant difference was observed in I K1 density among the regional cell types. The results indicate that M cells in humans, as in canines, show the greatest APD and that a gradient of I to1 density is present in the transmural ventricular wall. Therefore, the human right ventricle shows significant transmural heterogeneity in AP morphology and I to1properties.

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NP1 Protein of the Bocaparvovirus Minute Virus of Canines Controls Access to the Viral Capsid Genes via Its Role in RNA Processing

Journal of Virology ◽

10.1128/jvi.02618-15 ◽

2015 ◽

Vol 90 (4) ◽

pp. 1718-1728 ◽

Cited By ~ 19

Author(s):

Olufemi O. Fasina ◽

Yanming Dong ◽

David J. Pintel

Keyword(s):

Rna Processing ◽

Alternative Polyadenylation ◽

Genome Replication ◽

Coding Region ◽

Content Type ◽

Mrna Transcripts ◽

Viral Genome Replication ◽

Minute Virus ◽

Polyadenylation Sites ◽

The Right

ABSTRACTMinute virus of canines (MVC) is an autonomous parvovirus in the genusBocaparvovirus. It has a single promoter that generates a single pre-mRNA processed via alternative splicing and alternative polyadenylation to produce at least 8 mRNA transcripts. MVC contains two polyadenylation sites, one at the right-hand end of the genome, (pA)d, and another complex site, (pA)p, within the capsid-coding region. During viral infection, the mRNAs must extend through (pA)p and undergo additional splicing of the immediately upstream 3D∕3A intron to access the capsid gene. MVC NP1 is a 22-kDa nuclear phosphoprotein unique to the genusBocaparvovirusof theParvovirinaewhich we have shown governs suppression of (pA)p independently of viral genome replication. We show here that in addition to suppression of (pA)p, NP1 is also required for the excision of the MVC 3D∕3A intron, independently of its effect on alternative polyadenylation. Mutations of the arginine∕serine (SR) di-repeats within the intrinsically disordered amino terminus of NP1 are required for splicing of the capsid transcript but not suppression of polyadenylation at (pA)p. 3′-end processing of MVC mRNAs at (pA)p is critical for viral genome replication and the optimal expression of NP1 and NS1. Thus, a finely tuned balance between (pA)p suppression and usage is necessary for efficient virus replication. NP1 is the first parvovirus protein implicated in RNA processing. Its characterization reveals another way that parvoviruses govern access to their capsid protein genes, namely, at the RNA level, by regulating the essential splicing of an intron and the suppression of an internal polyadenylation site.IMPORTANCETheParvovirinaeare small nonenveloped icosahedral viruses that are important pathogens in many animal species, including humans. Although parvoviruses have only subtle early-to-late expression shifts, they all regulate access to their capsid genes. Minute virus of canines (MVC) is an autonomous parvovirus in the genusBocaparvovirus. It has a single promoter generating a single pre-mRNA which is processed via alternative splicing and alternative polyadenylation to generate at least 8 mRNA transcripts. MVC contains two polyadenylation sites, one at the right-hand end of the genome, (pA)d, and another, (pA)p, within the capsid-coding region. It had not been clear how the potent internal polyadenylation motif is suppressed to allow processing, export, and accumulation of the spliced capsid protein-encoding mRNAs. We show here that MVC NP1, the first parvovirus protein to be implicated in RNA processing, governs access to the MVC capsid gene by facilitating splicing and suppressing internal polyadenylation of MVC pre-mRNAs.

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Emerging Roles of RNA 3′-end Cleavage and Polyadenylation in Pathogenesis, Diagnosis and Therapy of Human Disorders

Biomolecules ◽

10.3390/biom10060915 ◽

2020 ◽

Vol 10 (6) ◽

pp. 915 ◽

Cited By ~ 3

Author(s):

Jamie Nourse ◽

Stefano Spada ◽

Sven Danckwardt

Keyword(s):

Rna Processing ◽

Therapeutic Potential ◽

Protein Complexes ◽

Alternative Polyadenylation ◽

Rna Motifs ◽

Physiological Processes ◽

Human Disorders ◽

Tightly Coupled ◽

Cleavage And Polyadenylation ◽

Health And Disease

A crucial feature of gene expression involves RNA processing to produce 3′ ends through a process termed 3′ end cleavage and polyadenylation (CPA). This ensures the nascent RNA molecule can exit the nucleus and be translated to ultimately give rise to a protein which can execute a function. Further, alternative polyadenylation (APA) can produce distinct transcript isoforms, profoundly expanding the complexity of the transcriptome. CPA is carried out by multi-component protein complexes interacting with multiple RNA motifs and is tightly coupled to transcription, other steps of RNA processing, and even epigenetic modifications. CPA and APA contribute to the maintenance of a multitude of diverse physiological processes. It is therefore not surprising that disruptions of CPA and APA can lead to devastating disorders. Here, we review potential CPA and APA mechanisms involving both loss and gain of function that can have tremendous impacts on health and disease. Ultimately we highlight the emerging diagnostic and therapeutic potential CPA and APA offer.

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RBBP6 activates the pre-mRNA 3′-end processing machinery in humans

10.1101/2021.11.02.466915 ◽

2021 ◽

Author(s):

Vytaute Boreikaite ◽

Thomas Elliot ◽

Jason Chin ◽

Lori A Passmore

Keyword(s):

Rna Processing ◽

Mrna Localization ◽

Dependent Manner ◽

Endonucleolytic Cleavage ◽

Mature Transcript ◽

Cleavage And Polyadenylation ◽

Specificity Factor ◽

Additional Protein ◽

Stimulatory Factor

3′-end processing of most human mRNAs is carried out by the cleavage and polyadenylation specificity factor (CPSF; CPF in yeast). Endonucleolytic cleavage of the nascent pre-mRNA defines the 3′-end of the mature transcript, which is important for mRNA localization, translation and stability. Cleavage must therefore be tightly regulated. Here, we reconstitute specific and efficient 3′-endonuclease activity of human CPSF with purified proteins. This requires the seven-subunit CPSF as well as three additional protein factors: cleavage stimulatory factor (CStF), cleavage factor IIm (CFIIm) and, importantly, the multi-domain protein RBBP6. Unlike its yeast homologue Mpe1, which is a stable subunit of CPF, RBBP6 does not copurify with CPSF and is recruited in an RNA-dependent manner. Sequence and mutational analyses suggest that RBBP6 interacts with the WDR33 and CPSF73 subunits of CPSF. Thus, it is likely that the role of RBBP6 is conserved from yeast to human. Overall, our data are consistent with CPSF endonuclease activation and site-specific pre-mRNA cleavage being highly controlled to maintain fidelity in RNA processing.

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A GATA-dependent right ventricular enhancer controls dHAND transcription in the developing heart

Development ◽

10.1242/dev.127.24.5331 ◽

2000 ◽

Vol 127 (24) ◽

pp. 5331-5341 ◽

Cited By ~ 2

Author(s):

D.G. McFadden ◽

J. Charite ◽

J.A. Richardson ◽

D. Srivastava ◽

A.B. Firulli ◽

...

Keyword(s):

Right Ventricle ◽

Right Ventricular ◽

Neural Crest ◽

Regulatory Elements ◽

Specific Gene ◽

Upstream Sequence ◽

Cardiac Progenitors ◽

Gata Factors ◽

Developing Heart ◽

The Right

Heart formation in vertebrates is believed to occur in a segmental fashion, with discreet populations of cardiac progenitors giving rise to different chambers of the heart. However, the mechanisms involved in specification of different chamber lineages are unclear. The basic helix-loop-helix transcription factor dHAND is expressed in cardiac precursors throughout the cardiac crescent and the linear heart tube, before becoming restricted to the right ventricular chamber at the onset of looping morphogenesis. dHAND is also expressed in the branchial arch neural crest, which contributes to craniofacial structures and the aortic arch arteries. Using a series of dHAND-lacZ reporter genes in transgenic mice, we show that cardiac and neural crest expression of dHAND are controlled by separate upstream enhancers and we describe a composite cardiac-specific enhancer that directs lacZ expression in a pattern that mimics that of the endogenous dHAND gene throughout heart development. Deletion analysis reduced this enhancer to a 1.5 kb region and identified subregions responsible for expression in the right ventricle and cardiac outflow tract. Comparison of mouse regulatory elements required for right ventricular expression to the human dHAND upstream sequence revealed two conserved consensus sites for binding of GATA transcription factors. Mutation of these sites abolished transgene expression in the right ventricle, identifying dHAND as a direct transcriptional target of GATA factors during right ventricle development. Since GATA factors are not chamber-restricted, these findings suggest the existence of positive and/or negative coregulators that cooperate with GATA factors to control right ventricular-specific gene expression in the developing heart.

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The Human Bocavirus 1 NP1 Protein Is a Multifunctional Regulator of Viral RNA Processing

Journal of Virology ◽

10.1128/jvi.01187-18 ◽

2018 ◽

Vol 92 (22) ◽

Cited By ~ 3

Author(s):

Yanming Dong ◽

Olufemi O. Fasina ◽

David J. Pintel

Keyword(s):

Rna Processing ◽

Alternative Polyadenylation ◽

Specific Protein ◽

Transcription Unit ◽

Capsid Gene ◽

Human Bocavirus ◽

Content Type ◽

Cleavage And Polyadenylation ◽

Minute Virus

ABSTRACTHuman bocavirus 1 (HBoV1) encodes a genus-specific protein, NP1, which regulates viral alternative pre-mRNA processing. Similar to NP1 of the related bocavirus minute virus of canine (MVC), HBoV1 NP1 suppressed cleavage and polyadenylation of RNAs at the viral internal polyadenylation site (pA)p. HBoV1 (pA)p is a complex region. It contains 5 significant cleavage and polyadenylation sites, and NP1 was found to regulate only the three of these sites that are governed by canonical AAUAAA hexamer signals. HBoV1 NP1 also facilitated splicing of the upstream intron adjacent to (pA)p. Alternative polyadenylation and splicing of the upstream intron were independent of each other, functioned efficiently within an isolated transcription unit, and were responsive independent of NP1. Characterization of HBoV1 NP1 generalizes its function within the genusBocaparvovirus, uncovers important differences, and provides important comparisons with MVC NP1 for mechanistic and evolutionary considerations.IMPORTANCETheParvovirinaeare small nonenveloped icosahedral viruses that are important pathogens in many animal species, including humans. The NP1 protein of human bocavirus 1 (HBoV1), similar to NP1 of the bocavirus minute virus of canine (MVC), regulates viral alternative RNA processing by both suppressing polyadenylation at an internal site, (pA)p, and facilitating splicing of an upstream adjacent intron. These effects allow both extension into the capsid gene and splicing of the viral pre-mRNA that correctly registers the capsid gene open reading frame. Characterization of HBoV1 NP1 generalizes this central mode of parvovirus gene regulation to another member of the bocavirus genus and uncovers both important similarities and differences in function compared to MVC NP1 that will be important for future comparative studies.

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