scholarly journals Structural insights into the assembly and polyA signal recognition mechanism of the human CPSF complex

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Marcello Clerici ◽  
Marco Faini ◽  
Ruedi Aebersold ◽  
Martin Jinek
Keyword(s):  
Mammalian Cells ◽  
Rna Binding ◽  
Molecular Architecture ◽  
Binding Assays ◽  
Critical Determinant ◽  
Recognition Mechanism ◽  
Cleavage And Polyadenylation ◽  
Specificity Factor ◽  
Motif Recognition ◽  
Mrna Polyadenylation

3’ polyadenylation is a key step in eukaryotic mRNA biogenesis. In mammalian cells, this process is dependent on the recognition of the hexanucleotide AAUAAA motif in the pre-mRNA polyadenylation signal by the cleavage and polyadenylation specificity factor (CPSF) complex. A core CPSF complex comprising CPSF160, WDR33, CPSF30 and Fip1 is sufficient for AAUAAA motif recognition, yet the molecular interactions underpinning its assembly and mechanism of PAS recognition are not understood. Based on cross-linking-coupled mass spectrometry, crystal structure of the CPSF160-WDR33 subcomplex and biochemical assays, we define the molecular architecture of the core human CPSF complex, identifying specific domains involved in inter-subunit interactions. In addition to zinc finger domains in CPSF30, we identify using quantitative RNA-binding assays an N-terminal lysine/arginine-rich motif in WDR33 as a critical determinant of specific AAUAAA motif recognition. Together, these results shed light on the function of CPSF in mediating PAS-dependent RNA cleavage and polyadenylation.

scholarly journals Cleavage and polyadenylation specificity factor 30: An RNA-binding zinc-finger protein with an unexpected 2Fe–2S cluster

2016 ◽  
Vol 113 (17) ◽  
pp. 4700-4705 ◽  
Author(s):  
Geoffrey D. Shimberg ◽  
Jamie L. Michalek ◽  
Abdulafeez A. Oluyadi ◽  
Andria V. Rodrigues ◽  
Beth E. Zucconi ◽  
...  
Keyword(s):  
Zinc Finger ◽  
Inductively Coupled Plasma ◽  
Rna Binding ◽  
Zinc Finger Protein ◽  
Mobility Shift ◽  
Inductively Coupled ◽  
Plasma Mass Spectrometry ◽  
Cleavage And Polyadenylation ◽  
Specificity Factor ◽  
Electrophoretic Mobility Shift Assays

Cleavage and polyadenylation specificity factor 30 (CPSF30) is a key protein involved in pre-mRNA processing. CPSF30 contains five Cys3His domains (annotated as “zinc-finger” domains). Using inductively coupled plasma mass spectrometry, X-ray absorption spectroscopy, and UV-visible spectroscopy, we report that CPSF30 is isolated with iron, in addition to zinc. Iron is present in CPSF30 as a 2Fe–2S cluster and uses one of the Cys3His domains; 2Fe–2S clusters with a Cys3His ligand set are rare and notably have also been identified in MitoNEET, a protein that was also annotated as a zinc finger. These findings support a role for iron in some zinc-finger proteins. Using electrophoretic mobility shift assays and fluorescence anisotropy, we report that CPSF30 selectively recognizes the AU-rich hexamer (AAUAAA) sequence present in pre-mRNA, providing the first molecular-based evidence to our knowledge for CPSF30/RNA binding. Removal of zinc, or both zinc and iron, abrogates binding, whereas removal of just iron significantly lessens binding. From these data we propose a model for RNA recognition that involves a metal-dependent cooperative binding mechanism.

scholarly journals A crystal structure of a collaborative RNA regulatory complex reveals mechanisms to refine target specificity

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Chen Qiu ◽  
Vandita D Bhat ◽  
Sanjana Rajeev ◽  
Chi Zhang ◽  
Alexa E Lasley ◽  
...  
Keyword(s):  
Crystal Structure ◽  
In Vitro Selection ◽  
Rna Binding ◽  
Sequence Specificity ◽  
Binding Assays ◽  
Regulatory Complex ◽  
Rna Motif ◽  
Rna Complex ◽  
Motif Recognition

In the Caenorhabditis elegans germline, fem-3 Binding Factor (FBF) partners with LST-1 to maintain stem cells. A crystal structure of an FBF-2/LST-1/RNA complex revealed that FBF-2 recognizes a short RNA motif different from the characteristic 9-nt FBF binding element, and compact motif recognition coincided with curvature changes in the FBF-2 scaffold. Previously, we engineered FBF-2 to favor recognition of shorter RNA motifs without curvature change (Bhat et al., 2019). In vitro selection of RNAs bound by FBF-2 suggested sequence specificity in the central region of the compact element. This bias, reflected in the crystal structure, was validated in RNA-binding assays. FBF-2 has the intrinsic ability to bind to this shorter motif. LST-1 weakens FBF-2 binding affinity for short and long motifs, which may increase target selectivity. Our findings highlight the role of FBF scaffold flexibility in RNA recognition and suggest a new mechanism by which protein partners refine target site selection.

scholarly journals RNA structure is a critical determinant of poly(A) site recognition by cleavage and polyadenylation specificity factor.

1996 ◽  
Vol 16 (9) ◽  
pp. 4942-4951 ◽  
Author(s):  
B R Graveley ◽  
E S Fleming ◽  
G M Gilmartin
Keyword(s):  
Human Immunodeficiency Virus ◽  
Rna Structure ◽  
Critical Determinant ◽  
Immunodeficiency Virus ◽  
Cleavage And Polyadenylation ◽  
Specificity Factor ◽  
A Site ◽  
Site Recognition

Sequence conservation among mammalian poly(A) sites is limited to the sequence AAUAAA, coupled with an amorphous downstream U- or GU-rich region. Since these sequences may also occur within the coding region of mRNAs, additional information must be required to define authentic poly(A) sites. Several poly(A) sites have been shown to contain sequences outside the core elements that enhance the efficiency of 3' processing in vivo and in vitro. The human immunodeficiency virus type 1, equine infectious anemia virus, and adenovirus L1 3' processing enhancers have been shown to promote the binding of cleavage and polyadenylation specificity factor (CPSF), the factor responsible for recognition of AAUAAA, to the pre-mRNA, thereby facilitating the assembly of a stable 3' processing complex. We have used in vitro selection to examine the mechanism by which the human immunodeficiency virus type 1 3' processing enhancer promotes the interaction of CPSF with the AAUAAA hexamer. Surprisingly, RNAs selected for efficient polyadenylation were related by structure rather than sequence. Therefore, in the absence of extensive sequence conservation, our results strongly suggest that RNA structure is a critical determinant of poly(A) site recognition by CPSF and may play a key role in poly(A) site definition.

scholarly journals Overlapping Local and Long-Range RNA-RNA Interactions Modulate Dengue Virus Genome Cyclization and Replication

2015 ◽  
Vol 89 (6) ◽  
pp. 3430-3437 ◽  
Author(s):  
Luana de Borba ◽  
Sergio M. Villordo ◽  
Nestor G. Iglesias ◽  
Claudia V. Filomatori ◽  
Leopoldo G. Gebhard ◽  
...  
Keyword(s):  
Dengue Virus ◽  
Mammalian Cells ◽  
Untranslated Region ◽  
Rna Binding ◽  
Rna Folding ◽  
Virus Genome ◽  
Binding Assays ◽  
Functional Studies ◽  
Chemical Probing ◽  
Dumbbell Structure

The dengue virus genome is a dynamic molecule that adopts different conformations in the infected cell. Here, using RNA folding predictions, chemical probing analysis, RNA binding assays, and functional studies, we identified newcis-acting elements present in the capsid coding sequence that facilitate cyclization of the viral RNA by hybridization with a sequence involved in a local dumbbell structure at the viral 3′ untranslated region (UTR). The identified interaction differentially enhances viral replication in mosquito and mammalian cells.

scholarly journals CPEB phosphorylation and cytoplasmic polyadenylation are catalyzed by the kinase IAK1/Eg2 in maturing mouse oocytes

Development ◽  
2001 ◽  
Vol 128 (14) ◽  
pp. 2815-2822 ◽  
Author(s):  
Rebecca Hodgman ◽  
Joyce Tay ◽  
Raul Mendez ◽  
Joel D. Richter
Keyword(s):  
Xenopus Oocytes ◽  
Rna Binding ◽  
Binding Protein ◽  
Dominant Negative ◽  
Cytoplasmic Polyadenylation ◽  
Mouse Oocytes ◽  
Dominant Negative Mutation ◽  
Murine Homologue ◽  
Cleavage And Polyadenylation ◽  
Specificity Factor

In both vertebrates and invertebrates, the expression of several maternal mRNAs is regulated by cytoplasmic polyadenylation. In Xenopus oocytes, where most of the biochemical details of this process have been examined, polyadenylation is controlled by CPEB, a sequence-specific RNA binding protein. The activity of CPEB, which is to recruit cleavage and polyadenylation specificity factor (CPSF) and poly(A) polymerase (PAP) into an active cytoplasmic polyadenylation complex, is controlled by Eg2-catalyzed phosphorylation. Soon after CPEB phosphorylation and resulting polyadenylation take place, the interaction between maskin, a CPEB-associated factor, and eIF4E, the cap-binding protein, is destroyed, which results in the recruitment of mRNA into polysomes. Polyadenylation also occurs in maturing mouse oocytes, although the biochemical events that govern the reaction in these cells are not known. In this study, we have examined the phosphorylation of CPEB and have assessed the necessity of this protein for polyadenylation in maturing mouse oocytes. Immunohistochemistry has revealed that all the factors that control polyadenylation and translation in Xenopus oocytes (CPEB, CPSF, PAP, maskin, and IAK1, the murine homologue of Eg2) are also present in the cytoplasm of mouse oocytes. After the induction of maturation, a kinase is activated that phosphorylates CPEB on a critical regulatory residue, an event that is essential for CPEB activity. A peptide that competitively inhibits the activity of IAK1/Eg2 blocks the progression of meiosis in injected oocytes. Finally, a CPEB protein that acts as a dominant negative mutation because it cannot be phosphorylated by IAK1/Eg2, prevents cytoplasmic polyadenylation. These data indicate that cytoplasmic polyadenylation in mouse oocytes is mediated by IAK1/Eg2-catalyzed phosphorylation of CPEB.

scholarly journals Epidermal progenitors suppress GRHL3-mediated differentiation through intronic polyadenylation promoted by CPSF-HNRNPA3 collaboration

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Xin Chen ◽  
Sarah M. Lloyd ◽  
Junghun Kweon ◽  
Giovanni M. Gamalong ◽  
Xiaomin Bao
Keyword(s):  
Rna Binding ◽  
Rna Binding Proteins ◽  
Somatic Tissue ◽  
Keratinocyte Differentiation ◽  
Regenerative Capacity ◽  
Site Specific ◽  
First Intron ◽  
Cleavage And Polyadenylation ◽  
Specificity Factor ◽  
Skin Epidermis

AbstractIn self-renewing somatic tissue such as skin epidermis, terminal differentiation genes must be suppressed in progenitors to sustain regenerative capacity. Here we show that hundreds of intronic polyadenylation (IpA) sites are differentially used during keratinocyte differentiation, which is accompanied by downregulation of the Cleavage and Polyadenylation Specificity Factor (CPSF) complex. Sustained CPSF expression in undifferentiated keratinocytes requires the contribution from the transcription factor MYC. In keratinocytes cultured in undifferentiation condition, CSPF knockdown induces premature differentiation and partially affects dynamically used IpA sites. These sites include an IpA site located in the first intron of the differentiation activator GRHL3. CRISPR knockout of GRHL3 IpA increased full-length GRHL3 mRNA expression. Using a targeted genetic screen, we identify that HNRNPA3 interacts with CPSF and enhances GRHL3 IpA. Our data suggest a model where the interaction between CPSF and RNA-binding proteins, such as HNRNPA3, promotes site-specific IpA and suppresses premature differentiation in progenitors.

scholarly journals Unique Features of Plant Cleavage and Polyadenylation Specificity Factor Revealed by Proteomic Studies

2009 ◽  
Vol 151 (3) ◽  
pp. 1546-1556 ◽  
Author(s):  
Hongwei Zhao ◽  
Denghui Xing ◽  
Qingshun Quinn Li
Keyword(s):  
Cleavage And Polyadenylation ◽  
Specificity Factor

scholarly journals Staufen1 is imported into the nucleolus via a bipartite nuclear localization signal and several modulatory determinants

2005 ◽  
Vol 393 (1) ◽  
pp. 245-254 ◽  
Author(s):  
Catherine Martel ◽  
Paolo Macchi ◽  
Luc Furic ◽  
Michael A. Kiebler ◽  
Luc Desgroseillers
Keyword(s):  
Nuclear Localization ◽  
Nuclear Localization Signal ◽  
Nuclear Export ◽  
Mammalian Cells ◽  
Rna Binding ◽  
Binding Activity ◽  
Nuclear Trafficking ◽  
Binding Domains ◽  
Bipartite Nuclear Localization Signal ◽  
Localization Signal

Mammalian Stau1 (Staufen1), a modular protein composed of several dsRBDs (double-stranded RNA-binding domains), is probably involved in mRNA localization. Although Stau1 is mostly described in association with the rough endoplasmic reticulum and ribosomes in the cytoplasm, recent studies suggest that it may transit through the nucleus/nucleolus. Using a sensitive yeast import assay, we show that Stau1 is actively imported into the nucleus through a newly identified bipartite nuclear localization signal. As in yeast, the bipartite nuclear localization signal is necessary for Stau1 nuclear import in mammalian cells. It is also required for Stau1 nucleolar trafficking. However, Stau1 nuclear transit seems to be regulated by mechanisms that involve cytoplasmic retention and/or facilitated nuclear export. Cytoplasmic retention is mainly achieved through the action of dsRBD3, with dsRBD2 playing a supporting role in this function. Similarly, dsRBD3, but not its RNA-binding activity, is critical for Stau1 nucleolar trafficking. The function of dsRBD3 is strengthened or stabilized by the presence of dsRBD4 but prevented by the interdomain between dsRBD2 and dsRBD3. Altogether, these results suggest that Stau1 nuclear trafficking is a highly regulated process involving several determinants. The presence of Stau1 in the nucleus/nucleolus suggests that it may be involved in ribonucleoprotein formation in the nucleus and/or in other nuclear functions not necessarily related to mRNA transport.

scholarly journals Multimerization of Hepatitis Delta Antigen Is a Critical Determinant of RNA Binding Specificity

2009 ◽  
Vol 84 (3) ◽  
pp. 1406-1413 ◽  
Author(s):  
Brian C. Lin ◽  
Dawn A. Defenbaugh ◽  
John L. Casey
Keyword(s):  
Conformational Changes ◽  
Rna Binding ◽  
Site Directed Mutagenesis ◽  
Minimum Length ◽  
Critical Determinant ◽  
Hepatitis Delta ◽  
Ribonucleoprotein Complex ◽  
Delta Antigen ◽  
Hepatitis Delta Antigen

ABSTRACT Hepatitis delta virus (HDV) RNA forms an unbranched rod structure that is associated with hepatitis delta antigen (HDAg) in cells replicating HDV. Previous in vitro binding experiments using bacterially expressed HDAg showed that the formation of a minimal ribonucleoprotein complex requires an HDV unbranched rod RNA of at least about 300 nucleotides (nt) and suggested that HDAg binds the RNA as a multimer of fixed size. The present study specifically examines the role of HDAg multimerization in the formation of the HDV ribonucleoprotein complex (RNP). Disruption of HDAg multimerization by site-directed mutagenesis was found to profoundly alter the nature of RNP formation. Mutant HDAg proteins defective for multimerization exhibited neither the 300-nt RNA size requirement for binding nor specificity for the unbranched rod structure. The results unambiguously demonstrate that HDAg binds HDV RNA as a multimer and that the HDAg multimer is formed prior to binding the RNA. RNP formation was found to be temperature dependent, which is consistent with conformational changes occurring on binding. Finally, analysis of RNPs constructed with unbranched rod RNAs successively longer than the minimum length indicated that multimeric binding is not limited to the first HDAg bound and that a minimum RNA length of between 604 and 714 nt is required for binding of a second multimer. The results confirm the previous proposal that HDAg binds as a large multimer and demonstrate that the multimer is a critical determinant of the structure of the HDV RNP.

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