Polyadenylation Factor Subunit Research Articles

354-OR: Attenuation of Obesity-Induced Vascular Inflammation by microRNA-485 in Endothelial Cells

Obesity induced endothelial dysfunction contributes to the vascular inflammation. MicroRNAs (miRNAs) are implicated in the epigenetic regulation of inflammatory pathways. However, relevant miRNAs in the microvasculature, and their relation to chronic inflammation and metabolic dysfunction are poorly understood. Using micro-RNA Seq approach to profile microRNAs from the endothelial cells (ECs) of brown adipose tissue (BAT) of mice with diet-induced obesity (DIO), we identified microRNA- 485-5p (miR-485-5p) as one of the most differentially regulated miRNA. MiR-485-5p was rapidly induced in ECs in response to TNFα, highly enriched in the BAT vascular endothelium of mice with DIO, and was increased by 1.97 fold in human plasma samples with obesity. MiR-485-5p inhibition decreased TNF-α-induced NF-kB-responsive adhesion molecules VCAM-1 (32.6%) and ICAM-1 (34.7%) and phosphorylation of IKBα in ECs in vitro. Gain and loss-of-function studies revealed that neutralization of miR-485-5p potently inhibited leukocyte adhesion to EC monolayers by 20.8% while the overexpression had the opposite effects. Mechanistically, miR-485-5p inhibited the expression of its target gene PCF11 (~40.3%), a polyadenylation factor subunit, an effect that increased VCAM-1 (2.51 fold) and ICAM-1 (1.43 fold) expression as well as leukocyte adhesion to EC monolayers by 18.6% in vitro. Systemic neutralization of miR-485-5p in mice decreased leukocyte adhesion and rolling by 55.1% and 46.7% respectively by intravital microscopy. Finally, systemic delivery of LNA-anti-miR-485-5p inhibitor resulted in improved glucose and insulin tolerance and energy expenditure in mice with DIO while decreasing Ly6Chigh proinflammatory monocytes in blood as measured by FACS. Our findings indicate that miR-485-5p plays a key regulatory role in controlling vascular inflammation in diet-induced obesity, an effect that could be exploited for therapeutic intervention in obesity-induced chronic inflammation. Disclosure F.Bestepe: None. P.F.O'tierney-ginn: None. P.Alcaide: None. I.Z.Jaffe: None. B.Icli: None. R.Pal-ghosh: None. C.Fritsche: None. K.Lakhotiya: None. S.Smolgovsky: None. J.Weston: None. A.Stepanian: None. F.Alvarado: None. P.Catalano: None. Funding American Diabetes Association (1-16-JDF-046 to B.I.); National Institutes of Health (HL149999)

Genetic screen for suppression of transcriptional interference identifies a gain-of-function mutation in Pol2 termination factor Seb1

The system of long noncoding RNA (lncRNA)-mediated transcriptional interference that represses fission yeast phosphate homoeostasis gene pho1 provides a sensitive readout of genetic influences on cotranscriptional 3'-processing and termination and a tool for discovery of regulators of this phase of the Pol2 transcription cycle. Here, we conducted a genetic screen for relief of transcriptional interference that unveiled a mechanism by which Pol2 termination is enhanced via a gain-of-function mutation, G476S, in the RNA-binding domain of an essential termination factor, Seb1. The genetic and physical evidence for gain-of-function is compelling: 1) seb1-G476S de-represses pho1 and tgp1, both of which are subject to lncRNA-mediated transcriptional interference; 2) seb1-G476S elicits precocious lncRNA transcription termination in response to lncRNA 5'-proximal poly(A) signals; 3) seb1-G476S derepression of pho1 is effaced by loss-of-function mutations in cleavage and polyadenylation factor (CPF) subunits and termination factor Rhn1; 4) synthetic lethality of seb1-G476S with pho1 derepressive mutants rpb1-CTD-S7A and aps1∆ is rescued by CPF/Rhn1 loss-of-function alleles; and 5) seb1-G476S elicits an upstream shift in poly(A) site preference in several messenger RNA genes. A crystal structure of the Seb1-G476S RNA-binding domain indicates potential for gain of contacts from Ser476 to RNA nucleobases. To our knowledge, this is a unique instance of a gain-of-function phenotype in a eukaryal transcription termination protein.

Structure-function analysis of fission yeast cleavage and polyadenylation factor (CPF) subunit Ppn1 and its interactions with Dis2 and Swd22.

Fission yeast Cleavage and Polyadenylation Factor (CPF), a 13-subunit complex, executes the cotranscriptional 3’ processing of RNA polymerase II (Pol2) transcripts that precedes transcription termination. The three-subunit DPS sub-complex of CPF, consisting of a PP1-type phosphoprotein phosphatase Dis2, a WD-repeat protein Swd22, and a putative phosphatase regulatory factor Ppn1, associates with the CPF core to form the holo-CPF assembly. Here we probed the functional, physical, and genetic interactions of DPS by focusing on the Ppn1 subunit, which mediates association of DPS with the core. Transcriptional profiling by RNA-seq defined limited but highly concordant sets of protein-coding genes that were dysregulated in ppn1Δ, swd22Δ and dis2Δ cells, which included the DPSΔ down-regulated phosphate homeostasis genes pho1 and pho84 that are controlled by lncRNA-mediated transcriptional interference. Essential and inessential modules of the 710-aa Ppn1 protein were defined by testing the effects of Ppn1 truncations in multiple genetic backgrounds in which Ppn1 is required for growth. An N-terminal 172-aa disordered region was dispensable and its deletion alleviated hypomorphic phenotypes caused by deleting C-terminal aa 640–710. A TFIIS-like domain (aa 173–330) was not required for viability but was important for Ppn1 activity in phosphate homeostasis. Distinct sites within Ppn1 for binding to Dis2 (spanning Ppn1 aa 506 to 532) and Swd22 (from Ppn1 aa 533 to 578) were demarcated by yeast two-hybrid assays. Dis2 interaction-defective missense mutants of full-length Ppn1 (that retained Swd22 interaction) were employed to show that binding to Dis2 (or its paralog Sds21) was necessary for Ppn1 biological activity. Ppn1 function was severely compromised by missense mutations that selectively affected its binding to Swd22.

Transcriptional profiling of fission yeast RNA polymerase II CTD mutants.

The carboxyl-terminal domain (CTD) of RNA polymerase II consists of tandem repeats of heptapeptide Y1S2P3T4S5P6S7. The CTD recruits proteins that drive or regulate gene expression. The trafficking of CTD-interacting proteins is orchestrated by remodeling CTD primary structure via Ser/Thr/Tyr phosphorylation and proline cis-trans isomerization, which collectively inscribe a CTD code. The fission yeast CTD consists of 29 repeats. To decipher the output of the fission yeast CTD code, we manipulated CTD length and amino acid content and gauged effects of these changes on gene expression. Whereas deleting 11 heptads has no effect on yeast growth, RNA-seq revealed that 25% of protein-coding transcripts were dysregulated. We profiled the transcriptomes of full-length CTD mutants, in which: all Tyr1 residues were replaced by Phe; all Ser2, Thr4, or Ser7 positions were changed to Ala; and half of the essential CTD code “letters” Pro3, Ser5, and Pro6 were mutated to Ala. Overlapping RNA-seq profiles suggested that a quarter of the up-regulated mRNAs and half of the down-regulated mRNAs seen in full-length CTD mutants might be attributable to a decrement in wild-type heptad number. Concordant mutant-specific profiles were observed for Y1F, S2A, and T4A cells, and for P6•P6A and S5•S5A cells, suggesting that Tyr1–Ser2–Thr4 and Ser5–Pro6 comprise distinct “words” in the CTD code. The phosphate regulon, which is repressed by lncRNA-mediated transcription interference, is de-repressed by CTD mutations P6•P6A and S5•S5A. De-repression of pho1 in P6•P6A and S5•S5A cells depends on cleavage and polyadenylation factor subunits Swd22 and Ppn1 and termination factor Rhn1, signifying that Pro6 and Ser5 mutations elicit precocious lncRNA 3′-processing/termination.

Fission yeast Ctf1, a cleavage and polyadenylation factor subunit is required for the maintenance of genomic integrity.

Accurate segregation of chromosome during mitosis requires the coordinated action of several cell cycle checkpoints that monitor replication of the genome and the attachment of sister chromatids to the mitotic spindle apparatus. Here we have characterized the fission yeast Ctf1, an ortholog of S. cerevisiae Rna15 in the maintenance of genomic integrity. The ctf1 is nonessential for the cell survival and its deletion strain exhibit cold sensitivity. The ctf1 deleted cells exhibit genetic interaction with spindle checkpoint protein Mad2 and Bub1. The deletion of ctf1 gene affects the chromosomal attachment to the mitotic spindle leading to the accumulation of Bub1-GFP foci. Ctf1 localizes to the nucleus and physically interacts with Rna14, a cleavage and polyadenylation factor.

Transient expression using agroinfiltration to study polyadenylation in plants.

An important facet of the study of polyadenylation in plants entails the use of transient expression to characterize poly(A) sites and the subunits of the polyadenylation complex. In this report, a simple and adaptable approach to transient expression in Nicotiana benthamiana leaves is described. This approach has been used to evaluate protein-protein interactions amongst different plant polyadenylation factor subunits and to study the usage and behavior of individual poly(A) sites from plant genes.

The Polyadenylation Factor Subunit CLEAVAGE AND POLYADENYLATION SPECIFICITY FACTOR30: A Key Factor of Programmed Cell Death and a Regulator of Immunity in Arabidopsis

Programmed cell death (PCD) is essential for several aspects of plant life, including development and stress responses. Indeed, incompatible plant-pathogen interactions are well known to induce the hypersensitive response, a localized cell death. Mutational analyses have identified several key PCD components, and we recently identified the mips1 mutant of Arabidopsis (Arabidopsis thaliana), which is deficient for the key enzyme catalyzing the limiting step of myoinositol synthesis. One of the most striking features of mips1 is the light-dependent formation of lesions on leaves due to salicylic acid (SA)-dependent PCD, revealing roles for myoinositol or inositol derivatives in the regulation of PCD. Here, we identified a regulator of plant PCD by screening for mutants that display transcriptomic profiles opposing that of the mips1 mutant. Our screen identified the oxt6 mutant, which has been described previously as being tolerant to oxidative stress. In the oxt6 mutant, a transfer DNA is inserted in the CLEAVAGE AND POLYADENYLATION SPECIFICITY FACTOR30 (CPSF30) gene, which encodes a polyadenylation factor subunit homolog. We show that CPSF30 is required for lesion formation in mips1 via SA-dependent signaling, that the prodeath function of CPSF30 is not mediated by changes in the glutathione status, and that CPSF30 activity is required for Pseudomonas syringae resistance. We also show that the oxt6 mutation suppresses cell death in other lesion-mimic mutants, including lesion-simulating disease1, mitogen-activated protein kinase4, constitutive expressor of pathogenesis-related genes5, and catalase2, suggesting that CPSF30 and, thus, the control of messenger RNA 3' end processing, through the regulation of SA production, is a key component of plant immune responses.

Distinctive interactions of the Arabidopsis homolog of the 30 kD subunit of the cleavage and polyadenylation specificity factor (AtCPSF30) with other polyadenylation factor subunits

BackgroundThe Arabidopsis ortholog of the 30 kD subunit of the mammalian Cleavage and Polyadenylation Specificity Factor (AtCPSF30) is an RNA-binding endonuclease that is associated with other Arabidopsis CPSF subunits (orthologs of the 160, 100, and 73 kD subunits of CPSF). In order to further explore the functions of AtCPSF30, the subcellular distribution of the protein was examined by over-expressing fusion proteins containing fluorescent reporters linked to different CPSF subunits.ResultsIt was found that AtCPSF30 by itself localizes, not to the nucleus, but to the cytoplasm. AtCPSF30 could be found in the nucleus when co-expressed with AtCPSF160 or AtCPSF73(I), one of the two Arabidopsis orthologs of CPSF73. This re-directing of AtCPSF30 indicates that AtCPSF30 is retained in the nucleus via interactions with either or both of these other CPSF subunits. Co-expression of AtCSPF30 with AtCPSF100 altered the location, not of AtCPSF30, but rather of AtCPSF100, with these proteins residing in the cytoplasm. Deletion of plant-specific N- or C-terminal domains of AtCPSF30 abolished various of the interactions between AtCPSF30 and other CPSF subunits, suggesting that the plant CPSF complex assembles via novel protein-protein interactions.ConclusionThese results suggest that the nuclear CPSF complex in plants is a dynamic one, and that the interactions between AtCPSF30 and other CPSF subunits are different from those existing in other eukaryotes.

A Polyadenylation Factor Subunit Implicated in Regulating Oxidative Signaling in Arabidopsis thaliana

BackgroundPlants respond to many unfavorable environmental conditions via signaling mediated by altered levels of various reactive oxygen species (ROS). To gain additional insight into oxidative signaling responses, Arabidopsis mutants that exhibited tolerance to oxidative stress were isolated. We describe herein the isolation and characterization of one such mutant, oxt6.Methodology/Principal FindingsThe oxt6 mutation is due to the disruption of a complex gene (At1g30460) that encodes the Arabidopsis ortholog of the 30-kD subunit of the cleavage and polyadenylation specificity factor (CPSF30) as well as a larger, related 65-kD protein. Expression of mRNAs encoding Arabidopsis CPSF30 alone was able to restore wild-type growth and stress susceptibility to the oxt6 mutant. Transcriptional profiling and single gene expression studies show elevated constitutive expression of a subset of genes that encode proteins containing thioredoxin- and glutaredoxin- related domains in the oxt6 mutant, suggesting that stress can be ameliorated by these gene classes. Bulk poly(A) tail length was not seemingly affected in the oxt6 mutant, but poly(A) site selection was different, indicating a subtle effect on polyadenylation in the mutant.Conclusions/SignificanceThese results implicate the Arabidopsis CPSF30 protein in the posttranscriptional control of the responses of plants to stress, and in particular to the expression of a set of genes that suffices to confer tolerance to oxidative stress.

Redox and heavy metal effects on the biochemical activities of an Arabidopsis polyadenylation factor subunit

The Arabidopsis CPSF30 ortholog (AtCPSF30) is an RNA-binding endonuclease that is part of the plant polyadenylation complex. Previous work (B. Addepalli, A.G. Hunt, Nucleic Acids Res. 35 (2007) 4453–4463) demonstrated that different zinc finger motifs in the protein were responsible for RNA-binding and nuclease activity, respectively. In this study, a more detailed functional map of AtCPSF30 is presented, a map that includes descriptions of novel biochemical activities. Elevated temperatures, the specific zinc chelator 1,10-phenanthroline, and the sulfhydryl reagent dithiothreitol all had differential inhibitory effects on the RNA-binding and nuclease activities. The endonuclease activity of AtCPSF30 was inhibited by relatively high (>100μM) concentrations of zinc, and this inhibition required a plant-specific N-terminal domain apart from the zinc finger core of the protein. ATP stimulated the nuclease activity in the presence of zinc, and this stimulation required a plant-specific C-terminal domain, again apart from the zinc finger core. These studies reveal a subtle and unexpected complexity to AtCPSF30, and raise the possibility that multiple avenues of regulation may impinge on this protein through different functional domains.

The Interaction Between Two Arabidopsis Polyadenylation Factor Subunits Involves an Evolutionarily-Conserved Motif and Has Implications for the Assembly and Function of the Polyadenylation Complex

The polyadenylation factor subunit "Factor Interacting with Poly(A) polymerase" (Fip1) is an important bridging subunit in the eukaryotic polyadenylation complex. To better understand the functioning of Fip1 in Arabidopsis, a random combinatorial screen for peptides that interact with a conserved plant-specific domain in the protein was conducted. A search of the Arabidopsis proteome using these Fip1-binding peptides as queries resulted in the identification of a number of putative Fip1-interacting proteins. One of these was the polyadenylation factor subunit, CstF77. This purported interaction was confirmed by yeast two-hybrid and in vitro assays. Mutation of the motif identified in the phage display screen eliminated the interaction, corroborating the results of the phage display screen. The domain of CstF77 that interacts with Fip1 lies at its extreme C-terminus and is distinct from the part of CstF77 that binds CstF64. Taken together, these results suggest that Fip1 is situated near CstF64 in the polyadenylation complex.

A novel endonuclease activity associated with the Arabidopsis ortholog of the 30-kDa subunit of cleavage and polyadenylation specificity factor

The polyadenylation of messenger RNAs is mediated by a multi-subunit complex that is conserved in eukaryotes. Among the most interesting of these proteins is the 30-kDa-subunit of the Cleavage and Polyadenylation Specificity Factor, or CPSF30. In this study, the Arabidopsis CPSF30 ortholog, AtCPSF30, is characterized. This protein possesses an unexpected endonucleolytic activity that is apparent as an ability to nick and degrade linear as well as circular single-stranded RNA. Endonucleolytic action by AtCPSF30 leaves RNA 3′ ends with hydroxyl groups, as they can be labeled by RNA ligase with [32P]-cytidine-3′,5′-bisphosphate. Mutations in the first of the three CCCH zinc finger motifs of the protein abolish RNA binding by AtCPSF30 but have no discernible effects on nuclease activity. In contrast, mutations in the third zinc finger motif eliminate the nuclease activity of the protein, and have a modest effect on RNA binding. The N-terminal domain of another Arabidopsis polyadenylation factor subunit, AtFip1(V), dramatically inhibits the nuclease activity of AtCPSF30 but has a slight negative effect on the RNA-binding activity of the protein. These results indicate that AtCPSF30 is a probable processing endonuclease, and that its action is coordinated through its interaction with Fip1.

Calmodulin Interacts with and Regulates the RNA-Binding Activity of an Arabidopsis Polyadenylation Factor Subunit

The Arabidopsis (Arabidopsis thaliana) gene that encodes the probable ortholog of the 30-kD subunit of the mammalian cleavage and polyadenylation specificity factor (CPSF) is a complex one, encoding small (approximately 28 kD) and large (approximately 68 kD) polypeptides. The small polypeptide (AtCPSF30) corresponds to CPSF30 and is the focus of this study. Recombinant AtCPSF30 was purified from Escherichia coli and found to possess RNA-binding activity. Mutational analysis indicated that an evolutionarily conserved central core of AtCPSF30 is involved in RNA binding, but that RNA binding also requires a short sequence adjacent to the N terminus of the central core. AtCPSF30 was found to bind calmodulin, and calmodulin inhibited the RNA-binding activity of the protein in a calcium-dependent manner. Mutational analysis showed that a small part of the protein, again adjacent to the N terminus of the conserved core, is responsible for calmodulin binding; point mutations in this region abolished both binding to and inhibition of RNA binding by calmodulin. Interestingly, AtCPSF30 was capable of self-interactions. This property also mapped to the central conserved core of the protein. However, calmodulin had no discernible effect on the self-association. These results show that the central portion of AtCPSF30 is involved in a number of important functions, and they raise interesting possibilities for both the interplay between splicing and polyadenylation and the regulation of these processes by stimuli that act through calmodulin.

An Arabidopsis Fip1 Homolog Interacts with RNA and Provides Conceptual Links with a Number of Other Polyadenylation Factor Subunits

The protein Fip1 is an important subunit of the eukaryotic polyadenylation apparatus, since it provides a bridge of sorts between poly(A) polymerase, other subunits of the polyadenylation apparatus, and the substrate RNA. In this study, a previously unreported Arabidopsis Fip1 homolog is characterized. The gene for this protein resides on chromosome V and encodes a 1196-amino acid polypeptide. Yeast two-hybrid and in vitro assays indicate that the N-terminal 137 amino acids of the Arabidopsis Fip1 protein interact with poly(A) polymerase (PAP). This domain also stimulates the activity of the PAP. Interestingly, this part of the Arabidopsis Fip1 interacts with Arabidopsis homologs of CstF77, CPSF30, CFIm-25, and PabN1. The interactions with CstF77, CPSF30, and CFIm-25 are reminiscent in various respects of similar interactions seen in yeast and mammals, although the part of the Arabidopsis Fip1 protein that participates in these interactions has no apparent counterpart in other eukaryotic Fip1 proteins. Interactions between Fip1 and PabN1 have not been reported in other systems; this may represent plant-specific associations. The C-terminal 789 amino acids of the Arabidopsis Fip1 protein were found to contain an RNA-binding domain; this domain correlated with an intact arginine-rich region and had a marked preference for poly(G) among the four homopolymers studied. These results indicate that the Arabidopsis Fip1, like its human counterpart, is an RNA-binding protein. Moreover, they provide conceptual links between PAP and several other Arabidopsis polyadenylation factor subunit homologs.

The role of the yeast cleavage and polyadenylation factor subunit Ydh1p/Cft2p in pre-mRNA 3'-end formation.

Cleavage and polyadenylation factor (CPF) is a multi-protein complex that functions in pre-mRNA 3'-end formation and in the RNA polymerase II (RNAP II) transcription cycle. Ydh1p/Cft2p is an essential component of CPF but its precise role in 3'-end processing remained unclear. We found that mutations in YDH1 inhibited both the cleavage and the polyadenylation steps of the 3'-end formation reaction in vitro. Recently, we demonstrated that an important function of CPF lies in the recognition of poly(A) site sequences and RNA binding analyses suggesting that Ydh1p/Cft2p interacts with the poly(A) site region. Here we show that mutant ydh1 strains are deficient in the recognition of the ACT1 cleavage site in vivo. The C-terminal domain (CTD) of RNAP II plays a major role in coupling 3'-end processing and transcription. We provide evidence that Ydh1p/Cft2p interacts with the CTD of RNAP II, several other subunits of CPF and with Pcf11p, a component of CF IA. We propose that Ydh1p/Cft2p contributes to the formation of important interaction surfaces that mediate the dynamic association of CPF with RNAP II, the recognition of poly(A) site sequences and the assembly of the polyadenylation machinery on the RNA substrate.

An interaction between an Arabidopsis poly(A) polymerase and a homologue of the 100 kDa subunit of CPSF.

The Arabidopsis genome possesses a number of sequences that are predicted to encode proteins that are similar to mammalian and yeast polyadenylation factor subunits. One of these resides on chromosome V and has the potential to encode a polypeptide related to the 100 kDa subunit of the mammalian cleavage and polyadenylation specificity factor (CPSF). This gene encodes a ca. 2400 nucleotide mRNA that in turn can be translated to yield a polypeptide that is 39% identical to the mammalian CPSF100 protein. Antibodies raised against the Arabidopsis protein recognized distinctive polypeptides in nuclear extracts prepared from pea and wheat germ, consistent with the hypothesis that the Arabidopsis protein is resident in a nuclear polyadenylation complex. Interestingly, the Arabidopsis CPSF100 was found to interact with a portion of a nuclear poly(A) polymerase. This interaction was attributable to a 60 amino acid domain in the CPSF100 polypeptide and the N-terminal 220 amino acids of the poly(A) polymerase. An analogous interaction has yet to be described in other eukaryotes. The interaction with PAP thus indicates that the plant CPSF100 polypeptide is likely part of the 3'-end processing machinery, but suggests that this complex may function differently in plants than it does in mammals and yeast.

A polyadenylation factor subunit is the human homologue of the Drosophila suppressor of forked protein.

Polyadenylation of messenger RNA precursors is a complex process that requires multiple protein factors (for reviews, see refs 1, 2). Cleavage stimulation factor (CstF) is one of these, functioning together with cleavage-polyadenylation specificity factor, two cleavage factors, and poly(A)+ polymerase. CstF is composed of three subunits of M(r) 77, 64 and 50K. The 64K and 50K subunits contain, respectively, an RNP-type RNA-binding domain that contacts the pre-mRNA and transducin repeats characteristic of G-protein beta-subunits. Here we report the cloning and characterization of the 77K subunit of human CstF (referred to as 77K). We show that the 77K subunit is required for formation of active CstF and bridges the 64K and 50K subunits. Sequence analyses indicate that the 77K subunit is the homologue of the protein encoded by the Drosophila melanogaster suppressor of forked (su(f)) gene. Mutations in su(f) can enhance or suppress the effects of transposable element insertions, and our data indicate that this is due to changes in polyadenylation. Both the 77K subunit and the su(f) protein share homology with Saccharomyces cerevisiae RNA14, previously shown to be involved in mRNA metabolism. Our results thus also indicate that components of the complex polyadenylation machinery are conserved from yeast to man.