Publications by authors named "Clara L Kielkopf"

51 Publications

Solubilization of Expressed Proteins from Inclusion Bodies.

Cold Spring Harb Protoc 2021 Feb 1;2021(2):pdb.prot102210. Epub 2021 Feb 1.

The expression of foreign proteins at high levels in often results in the formation of cytoplasmic granules or inclusion bodies composed of insoluble aggregates of the expressed protein. These inclusion bodies can be seen with a phase-contrast microscope and are readily separated from most soluble and membrane-bound bacterial proteins, as described in this protocol.
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http://dx.doi.org/10.1101/pdb.prot102210DOI Listing
February 2021

Preparation of Cell Extracts for Purification of Soluble Proteins Expressed in .

Cold Spring Harb Protoc 2021 Feb 1;2021(2):pdb.prot102178. Epub 2021 Feb 1.

Recovery of intracellular proteins requires disruption of the host cell before the target protein is extracted and isolated. For cells enveloped in cell walls (such as ), vigorous methods are often required. This protocol focuses on lysis by sonication. Also included are methods for lysis by freeze-thaw and enzymatic treatments.
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http://dx.doi.org/10.1101/pdb.prot102178DOI Listing
February 2021

Subcellular Localization of Signal Peptide Fusion Proteins Expressed in .

Cold Spring Harb Protoc 2021 Feb 1;2021(2):pdb.prot102145. Epub 2021 Feb 1.

For expression of some proteins in , export to the periplasmic space is preferred over conventional expression in the cytosol. Export can be accomplished by fusing the coding sequence to DNA encoding a signal peptide (e.g., using pET-22b), which is cleaved by the bacterial signal peptidase as the protein is exported into the space between the inner and outer membranes of This protocol uses osmotic shock to release polypeptides from the periplasm. Although not quantitative, it should provide preliminary information on the cellular location of signal peptide fusion proteins.
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http://dx.doi.org/10.1101/pdb.prot102145DOI Listing
February 2021

Expression of Cloned Genes in Using IPTG-Inducible Promoters.

Cold Spring Harb Protoc 2021 Feb 1;2021(2):pdb.prot102137. Epub 2021 Feb 1.

Many expression vectors make use of the operon. In general, the operator () is located downstream from the promoter of the target gene, so that binding of the repressor blocks transcription initiation until lactose or the isopropyl-β-d-thiogalactopyranoside (IPTG) analog is added. The protocol given here is intended for use with IPTG-inducible vectors. l-Arabinose-inducible systems derived from the operon offer an alternative to expression systems based on the operon; guidance for their use is also provided.
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http://dx.doi.org/10.1101/pdb.prot102137DOI Listing
February 2021

Considerations for Membrane Protein Purification.

Cold Spring Harb Protoc 2021 Jan 4;2021(1):pdb.top102285. Epub 2021 Jan 4.

Isolating membrane proteins from their native cells while maintaining structural and functional integrity is challenging. Many detergents have been developed over the years that interact favorably with membrane proteins and mimic the physical properties of the lipid bilayer. Choosing the appropriate detergent is crucial for the successful extraction of a protein in its properly folded, active conformation.
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http://dx.doi.org/10.1101/pdb.top102285DOI Listing
January 2021

Preparation of Cell Extracts for Purification of Proteins Expressed in .

Cold Spring Harb Protoc 2021 Jan 4;2021(1):pdb.prot102186. Epub 2021 Jan 4.

Recovery of intracellular proteins requires disruption of the host cell before the target protein is extracted and isolated. Disruption methods vary depending on the type of cells, the total volume, and the number of samples being processed. For cells enveloped in cell walls (such as yeast), mild techniques such as hypotonic shock are not sufficient to achieve adequate lysis. More vigorous methods are often required. Although the preferred medium- or large-scale method of breaking yeast cells is mechanical shearing, lysis with the aid of glass beads in a BeadBeater is described here.
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http://dx.doi.org/10.1101/pdb.prot102186DOI Listing
January 2021

Expression of Cloned Genes in Using the Methanol-Inducible Promoter .

Cold Spring Harb Protoc 2021 Jan 4;2021(1):pdb.prot102160. Epub 2021 Jan 4.

is a methylotrophic yeast capable of metabolizing methanol as its sole carbon source. Growth in methanol-containing medium results in dramatic induction of genes in the alcohol oxidation pathway including alcohol oxidase (AOX), formaldehyde dehydrogenase (FLD), and dihydroxyacetone synthase (DHAS). These proteins may comprise up to 30% of the biomass. Investigators have exploited these methanol-dependent genes to generate tightly regulated expression vectors. Most vectors use the strong and tightly regulated promoter to drive heterologous protein expression. Obtaining integrated transformants requires more DNA than transformations into , where the gene is expressed from episomal plasmids; however, transformants are extremely stable and can be stored for many years.
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http://dx.doi.org/10.1101/pdb.prot102160DOI Listing
January 2021

Expressing Cloned Genes for Protein Production, Purification, and Analysis.

Cold Spring Harb Protoc 2021 Feb 1;2021(2):pdb.top102129. Epub 2021 Feb 1.

Obtaining high quantities of a specific protein directly from native sources is often challenging, particularly when dealing with human proteins. To overcome this obstacle, many researchers take advantage of heterologous expression systems by cloning genes into artificial vectors designed to operate within easily cultured cells, such as , (yeast), and several varieties of insect and mammalian cells. Heterologous expression systems also allow for easy modification of the protein to optimize expression, mutational analysis of specific sites within the protein and facilitate their purification with engineered affinity tags. Some degree of purification of the target protein is usually required for functional analysis. Purification to near homogeneity is essential for characterization of protein structure by X-ray crystallography or nuclear magnetic resonance (NMR) and characterization of the biochemical and biophysical properties of a protein, because contaminating proteins almost always adversely affect the results. Methods for producing and purifying proteins in several different expression platforms and using a variety of vectors are introduced here.
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http://dx.doi.org/10.1101/pdb.top102129DOI Listing
February 2021

Representative cancer-associated U2AF2 mutations alter RNA interactions and splicing.

J Biol Chem 2020 Dec 5;295(50):17148-17157. Epub 2020 Oct 5.

Center for RNA Biology, Department of Biochemistry and Biophysics, University of Rochester School of Medicine and Dentistry, Rochester, New York, USA

High-throughput sequencing of hematologic malignancies and other cancers has revealed recurrent mis-sense mutations of genes encoding pre-mRNA splicing factors. The essential splicing factor U2AF2 recognizes a polypyrimidine-tract splice-site signal and initiates spliceosome assembly. Here, we investigate representative, acquired mutations, namely N196K or G301D amino acid substitutions associated with leukemia or solid tumors, respectively. We determined crystal structures of the wild-type (WT) compared with N196K- or G301D-substituted U2AF2 proteins, each bound to a prototypical polypyrimidine tract, at 1.5, 1.4, or 1.7 Å resolutions. The N196K residue appears to stabilize the open conformation of U2AF2 with an inter-RNA recognition motif hydrogen bond, in agreement with an increased apparent RNA-binding affinity of the N196K-substituted protein. The G301D residue remains in a similar position as the WT residue, where unfavorable proximity to the RNA phosphodiester could explain the decreased RNA-binding affinity of the G301D-substituted protein. We found that expression of the G301D-substituted U2AF2 protein reduces splicing of a minigene transcript carrying prototypical splice sites. We further show that expression of either N196K- or G301D-substituted U2AF2 can subtly alter splicing of representative endogenous transcripts, despite the presence of endogenous, WT U2AF2 such as would be present in cancer cells. Altogether, our results demonstrate that acquired mutations such as N196K and G301D are capable of dysregulating gene expression for neoplastic transformation.
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http://dx.doi.org/10.1074/jbc.RA120.015339DOI Listing
December 2020

Purification of Fusion Proteins by Affinity Chromatography on Glutathione Resin.

Cold Spring Harb Protoc 2020 06 1;2020(6):102202. Epub 2020 Jun 1.

Fusion proteins that contain a glutathione -transferase (GST) moiety can be purified to near homogeneity by affinity chromatography on glutathione-linked resins. Glutathione immobilized on a chromatography matrix, such as agarose or Sepharose, acts as a substrate for the GST moiety of fusion proteins. Contaminating proteins are washed away, and the bound GST fusion proteins are then readily displaced from the resin by elution with buffers containing free glutathione.
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http://dx.doi.org/10.1101/pdb.prot102202DOI Listing
June 2020

Purification of Polyhistidine-Tagged Proteins by Immobilized Metal Affinity Chromatography.

Cold Spring Harb Protoc 2020 06 1;2020(6):102194. Epub 2020 Jun 1.

Immobilized metal affinity chromatography (IMAC) is based on the affinity of polyhistidine tracts for divalent metal cations (usually Ni) immobilized as transition metal chelate complexes on a chromatography resin. The main protocol here is optimized for use of Ni-NTA resin to purify soluble 6xHis-tagged proteins by a straightforward batch method during the binding step, followed by gravity flow for washes and elution. This protocol does not require any specialized equipment other than a simple glass or plastic column. IMAC resins can be used in multiple formats, including batch, gravity flow, centrifuge columns, and fast performance liquid chromatography (FPLC) systems. FPLC systems are designed specifically for the chromatographic separations of proteins and other biomolecules. These systems typically contain multiple pumps, an in-line UV absorption monitor, conductivity meter, pH meter, fraction collector, and other options that allow for the simultaneous purification, analysis, and fractionation of the sample. When linked with the appropriate instruments, an FPLC can become a high-precision, automated instrument that separates proteins at a high resolution. An alternative protocol is included here that describes 6xHis-tagged protein purification using FPLC. Procedures for the cleaning and regeneration of the IMAC resin for reuse are also described, and, finally, considerations for storing purified proteins are discussed.
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http://dx.doi.org/10.1101/pdb.prot102194DOI Listing
June 2020

Expression of Cloned Genes Using the Baculovirus Expression System.

Cold Spring Harb Protoc 2020 06 1;2020(6):102152. Epub 2020 Jun 1.

The general strategy of the baculovirus expression system is to infect insect cells with a virus that expresses a foreign protein at a very late stage of infection. Almost all baculovirus expression systems use the procedures for insect cell transfection, baculovirus production, and protein expression given in the main portion of this protocol. This protocol also includes a method that uses molecular biology techniques to produce recombinant baculovirus DNA in before transfection of insect cells. It is important to quantify the viral titer to achieve optimal and reproducible expression of target proteins. Accordingly, the viral plaque assay is also described here.
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http://dx.doi.org/10.1101/pdb.prot102152DOI Listing
June 2020

A splice site-sensing conformational switch in U2AF2 is modulated by U2AF1 and its recurrent myelodysplasia-associated mutation.

Nucleic Acids Res 2020 06;48(10):5695-5709

Department of Biochemistry and Biophysics and Center for RNA Biology, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642, USA.

An essential heterodimer of the U2AF1 and U2AF2 pre-mRNA splicing factors nucleates spliceosome assembly at polypyrimidine (Py) signals preceding the major class of 3' splice sites. U2AF1 frequently acquires an S34F-encoding mutation among patients with myelodysplastic syndromes (MDS). The influence of the U2AF1 subunit and its S34F mutation on the U2AF2 conformations remains unknown. Here, we employ single molecule Förster resonance energy transfer (FRET) to determine the influence of wild-type or S34F-substituted U2AF1 on the conformational dynamics of U2AF2 and its splice site RNA complexes. In the absence of RNA, the U2AF1 subunit stabilizes a high FRET value, which by structure-guided mutagenesis corresponds to a closed conformation of the tandem U2AF2 RNA recognition motifs (RRMs). When the U2AF heterodimer is bound to a strong, uridine-rich splice site, U2AF2 switches to a lower FRET value characteristic of an open, side-by-side arrangement of the RRMs. Remarkably, the U2AF heterodimer binds weak, uridine-poor Py tracts as a mixture of closed and open U2AF2 conformations, which are modulated by the S34F mutation. Shifts between open and closed U2AF2 may underlie U2AF1-dependent splicing of degenerate Py tracts and contribute to a subset of S34F-dysregulated splicing events in MDS patients.
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http://dx.doi.org/10.1093/nar/gkaa293DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7261175PMC
June 2020

Methods for Measuring the Concentrations of Proteins.

Cold Spring Harb Protoc 2020 04 1;2020(4):102277. Epub 2020 Apr 1.

Determining the concentration of protein samples generally is accomplished either by measuring the UV absorbance at 280 nm or by reacting the protein quantitatively with dyes and/or metal ions (Bradford, Lowry, or BCA assays). For purified proteins, UV absorbance remains the most popular method because it is fast, convenient, and reproducible; it does not consume the protein; and it requires no additional reagents, standards, or incubations. No method of protein concentration determination is perfect because each is subject to a different set of constraints such as interference of buffer components and contaminating proteins in direct UV determination () or reactivity of individual proteins and buffer components with the detecting reagents in colorimetric assays. In cases in which protein concentration is critical (e.g., determination of catalytic rate constants for an enzyme), it may be advisable to compare the results of several assays.
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http://dx.doi.org/10.1101/pdb.top102277DOI Listing
April 2020

Bradford Assay for Determining Protein Concentration.

Cold Spring Harb Protoc 2020 04 1;2020(4):102269. Epub 2020 Apr 1.

The Bradford assay is a quick and fairly sensitive method for measuring the concentrations of proteins. It is based on the shift in absorbance maximum of Coomassie Brilliant Blue G-250 dye from 465 to 595 nm following binding to denatured proteins in solution.
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http://dx.doi.org/10.1101/pdb.prot102269DOI Listing
April 2020

Structures of SF3b1 reveal a dynamic Achilles heel of spliceosome assembly: Implications for cancer-associated abnormalities and drug discovery.

Biochim Biophys Acta Gene Regul Mech 2019 Nov - Dec;1862(11-12):194440. Epub 2019 Nov 9.

Department of Biochemistry and Biophysics, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642, USA. Electronic address:

The pre-mRNA splicing factor SF3b1 exhibits recurrent mutations among hematologic malignancies and cancers, and consequently is a major therapeutic target of clinically-advanced spliceosome inhibitors. In this review, we highlight and rigorously analyze emerging views of SF3b1 conformational transitions, including the human SF3b particle either in isolation or bound to spliceosome inhibitors, and human or yeast spliceosome assemblies. Among spliceosome states characterized to date, an SF3b1 α-helical superhelix significantly closes to surround a U2 small nuclear RNA duplex with the pre-mRNA branch point sequence. The SF3b1 torus is locally unwound at an active site adenosine, whereas protein cofactors appear to stabilize overall closure in the spliceosome. Network analyses demonstrates that the natural SF3b1 dynamics mimic its conformational change in the spliceosome, raising the possibility of conformational selection underpinning spliceosome assembly. These dynamic SF3b1 conformations have consequences for gatekeeping of spliceosome assembly and therapeutic targeting of its cancer-associated dysfunction.
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http://dx.doi.org/10.1016/j.bbagrm.2019.194440DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6880240PMC
February 2020

Cus2 enforces the first ATP-dependent step of splicing by binding to yeast SF3b1 through a UHM-ULM interaction.

RNA 2019 08 20;25(8):1020-1037. Epub 2019 May 20.

Center for Molecular Biology of RNA, University of California, Santa Cruz, Santa Cruz, California 95064, USA.

Stable recognition of the intron branchpoint (BP) by the U2 snRNP to form the pre-spliceosome is the first ATP-dependent step of splicing. Genetic and biochemical data from yeast indicate that Cus2 aids U2 snRNA folding into the stem IIa conformation prior to pre-spliceosome formation. Cus2 must then be removed by an ATP-dependent function of Prp5 before assembly can progress. However, the location from which Cus2 is displaced and the nature of its binding to the U2 snRNP are unknown. Here, we show that Cus2 contains a conserved UHM (U2AF homology motif) that binds Hsh155, the yeast homolog of human SF3b1, through a conserved ULM (U2AF ligand motif). Mutations in either motif block binding and allow pre-spliceosome formation without ATP. A 2.0 Å resolution structure of the Hsh155 ULM in complex with the UHM of Tat-SF1, the human homolog of Cus2, and complementary binding assays show that the interaction is highly similar between yeast and humans. Furthermore, we show that Tat-SF1 can replace Cus2 function by enforcing ATP dependence of pre-spliceosome formation in yeast extracts. Cus2 is removed before pre-spliceosome formation, and both Cus2 and its Hsh155 ULM binding site are absent from available cryo-EM structure models. However, our data are consistent with the apparent location of the disordered Hsh155 ULM between the U2 stem-loop IIa and the HEAT repeats of Hsh155 that interact with Prp5. We propose a model in which Prp5 uses ATP to remove Cus2 from Hsh155 such that extended base-pairing between U2 snRNA and the intron BP can occur.
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http://dx.doi.org/10.1261/rna.070649.119DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6633205PMC
August 2019

Dynamic stacking of an expected branch point adenosine in duplexes containing pseudouridine-modified or unmodified U2 snRNA sites.

Biochem Biophys Res Commun 2019 04 21;511(2):416-421. Epub 2019 Feb 21.

Department of Biochemistry and Biophysics, Center for RNA Biology, University of Rochester School of Medicine and Dentistry, Rochester, NY, 14642, USA. Electronic address:

The pre-mRNA branch point sequence (BPS) anneals with a pseudouridine-modified region of the U2 small nuclear (sn)RNA, and offers a 2' hydroxyl group of a bulged adenosine as the nucleophile for the first catalytic step of pre-mRNA splicing. To increase our structural understanding of branch site selection, we characterized a duplex containing a BPS sequence that is common among multicellular eukaryotes (5'-UACUGAC-3') and the complementary U2 snRNA site using NMR. A major conformation of the expected branch site adenosine stacked within the duplex and paired with the conserved pseudouridine of the U2 snRNA strand. In contrast, the guanosine preceding the branch site appeared flexible and had weak contacts with the surrounding nucleotides. Pseudouridine-modified and unmodified U2 snRNA-BPS-containing duplexes remained structurally similar. These results highlight the importance of auxiliary factors to achieve the active bulged conformation of the branch site nucleophile for the first step of pre-mRNA splicing.
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http://dx.doi.org/10.1016/j.bbrc.2019.02.073DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6402984PMC
April 2019

The pre-mRNA splicing and transcription factor Tat-SF1 is a functional partner of the spliceosome SF3b1 subunit via a U2AF homology motif interface.

J Biol Chem 2019 02 19;294(8):2892-2902. Epub 2018 Dec 19.

From the Center for RNA Biology, Department of Biochemistry and Biophysics, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642

The transcription elongation and pre-mRNA splicing factor Tat-SF1 associates with the U2 small nuclear ribonucleoprotein (snRNP) of the spliceosome. However, the direct binding partner and underlying interactions mediating the Tat-SF1-U2 snRNP association remain unknown. Here, we identified SF3b1 as a Tat-SF1-interacting subunit of the U2 snRNP. Our 1.1 Å resolution crystal structure revealed that Tat-SF1 contains a U2AF homology motif (UHM) protein-protein interaction module. We demonstrated that Tat-SF1 preferentially and directly binds the SF3b1 subunit compared with other U2AF ligand motif (ULM)-containing splicing factors, and further established that SF3b1 association depends on the integrity of the Tat-SF1 UHM. We next compared the Tat-SF1-binding affinities for each of the five known SF3b1 ULMs and then determined the structures of representative high- and low-affinity SF3b1 ULM complexes with the Tat-SF1 UHM at 1.9 Å and 2.1 Å resolutions, respectively. These structures revealed a canonical UHM-ULM interface, comprising a Tat-SF1 binding pocket for a ULM tryptophan (SF3b1 Trp) and electrostatic interactions with a basic ULM tail. Importantly, we found that SF3b1 regulates Tat-SF1 levels and that these two factors influence expression of overlapping representative transcripts, consistent with a functional partnership of Tat-SF1 and SF3b1. Altogether, these results define a new molecular interface of the Tat-SF1-U2 snRNP complex for gene regulation.
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http://dx.doi.org/10.1074/jbc.RA118.006764DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6393619PMC
February 2019

Insights from structures of cancer-relevant pre-mRNA splicing factors.

Authors:
Clara L Kielkopf

Curr Opin Genet Dev 2018 02 10;48:57-66. Epub 2017 Nov 10.

Center for RNA Biology and Department of Biochemistry and Biophysics, University of Rochester School of Medicine and Dentistry, Rochester, NY, USA. Electronic address:

Pre-mRNA splicing factors recognize consensus signals within preliminary transcripts, and as cogs of the spliceosome machine, orchestrate the excision and rejoining of pre-mRNA regions for gene expression. Large-scale sequencing has demonstrated that mutations in key genes encoding pre-mRNA splicing factors are common among myeloid neoplasms and also occur in a variety of other cancers. This revelation offers new therapeutic opportunities to target pre-mRNA splicing vulnerabilities in hematologic and other malignancies. The mutated residues typically alter 3' splice site choice for a subset of transcripts. In this review, we highlight mechanistic insights from recent 3D structures that reveal the affected residues poised for pre-mRNA recognition.
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http://dx.doi.org/10.1016/j.gde.2017.10.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5871561PMC
February 2018

Cancer-Associated Mutations Mapped on High-Resolution Structures of the U2AF2 RNA Recognition Motifs.

Biochemistry 2017 09 1;56(36):4757-4761. Epub 2017 Sep 1.

Center for RNA Biology and Department of Biochemistry and Biophysics, University of Rochester School of Medicine and Dentistry , Rochester, New York 14642, United States.

Acquired point mutations of pre-mRNA splicing factors recur among cancers, leukemias, and related neoplasms. Several studies have established that somatic mutations of a U2AF1 subunit, which normally recognizes 3' splice site junctions, recur among myelodysplastic syndromes. The U2AF2 splicing factor recognizes polypyrimidine signals that precede most 3' splice sites as a heterodimer with U2AF1. In contrast with those of the well-studied U2AF1 subunit, descriptions of cancer-relevant U2AF2 mutations and their structural relationships are lacking. Here, we survey databases of cancer-associated mutations and identify recurring missense mutations in the U2AF2 gene. We determine ultra-high-resolution structures of the U2AF2 RNA recognition motifs (RRM1 and RRM2) at 1.1 Å resolution and map the structural locations of the mutated U2AF2 residues. Comparison with prior, lower-resolution structures of the tandem U2AF2 RRMs in the RNA-bound and apo states reveals clusters of cancer-associated mutations at the U2AF2 RRM-RNA or apo-RRM1-RRM2 interfaces. Although the role of U2AF2 mutations in malignant transformation remains uncertain, our results show that cancer-associated mutations correlate with functionally important surfaces of the U2AF2 splicing factor.
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http://dx.doi.org/10.1021/acs.biochem.7b00551DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6005654PMC
September 2017

Splicing Factor Mutations in Myelodysplasias: Insights from Spliceosome Structures.

Trends Genet 2017 05 31;33(5):336-348. Epub 2017 Mar 31.

Center for RNA Biology and Department of Biochemistry and Biophysics, University of Rochester School of Medicine and Dentistry, Rochester, NY, USA. Electronic address:

Somatic mutations of pre-mRNA splicing factors recur among patients with myelodysplastic syndrome (MDS) and related malignancies. Although these MDS-relevant mutations alter the splicing of a subset of transcripts, the mechanisms by which these single amino acid substitutions change gene expression remain controversial. New structures of spliceosome intermediates and associated protein complexes shed light on the molecular interactions mediated by 'hotspots' of the SF3B1 and U2AF1 pre-mRNA splicing factors. The frequently mutated SF3B1 residues contact the pre-mRNA splice site. Based on structural homology with other spliceosome subunits, and recent findings of altered RNA binding by mutant U2AF1 proteins, we suggest that affected U2AF1 residues also contact pre-mRNA. Altered pre-mRNA recognition emerges as a molecular theme among MDS-relevant mutations of pre-mRNA splicing factors.
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http://dx.doi.org/10.1016/j.tig.2017.03.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5447463PMC
May 2017

SF1 Phosphorylation Enhances Specific Binding to U2AF and Reduces Binding to 3'-Splice-Site RNA.

Biophys J 2016 Dec;111(12):2570-2586

Department of Biochemistry and Biophysics, University of Rochester School of Medicine, Rochester, New York. Electronic address:

Splicing factor 1 (SF1) recognizes 3' splice sites of the major class of introns as a ternary complex with U2AF and U2AF splicing factors. A conserved SPSP motif in a coiled-coil domain of SF1 is highly phosphorylated in proliferating human cells and is required for cell proliferation. The UHM kinase 1 (UHMK1), also called KIS, double-phosphorylates both serines of this SF1 motif. Here, we use isothermal titration calorimetry to demonstrate that UHMK1 phosphorylation of the SF1 SPSP motif slightly enhances specific binding of phospho-SF1 to its cognate U2AF protein partner. Conversely, quantitative fluorescence anisotropy RNA binding assays and isothermal titration calorimetry experiments establish that double-SPSP phosphorylation reduces phospho-SF1 and phospho-SF1-U2AF binding affinities for either optimal or suboptimal splice-site RNAs. Domain-substitution and mutagenesis experiments further demonstrate that arginines surrounding the phosphorylated SF1 loop are required for cooperative 3' splice site recognition by the SF1-U2AF complex (where cooperativity is defined as a nonadditive increase in RNA binding by the protein complex relative to the individual proteins). In the context of local, intracellular concentrations, the subtle effects of SF1 phosphorylation on its associations with U2AF and splice-site RNAs are likely to influence pre-mRNA splicing. However, considering roles for SF1 in pre-mRNA retention and transcriptional repression, as well as in splicing, future comprehensive investigations are needed to fully explain the requirement for SF1 SPSP phosphorylation in proliferating human cells.
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http://dx.doi.org/10.1016/j.bpj.2016.11.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5192697PMC
December 2016

Unmasking the U2AF homology motif family: a bona fide protein-protein interaction motif in disguise.

RNA 2016 12;22(12):1795-1807

Center for RNA Biology and Department for Biochemistry and Biophysics, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642, USA.

U2AF homology motifs (UHM) that recognize U2AF ligand motifs (ULM) are an emerging family of protein-protein interaction modules. UHM-ULM interactions recur in pre-mRNA splicing factors including U2AF1 and SF3b1, which are frequently mutated in myelodysplastic syndromes. The core topology of the UHM resembles an RNA recognition motif and is often mistakenly classified within this large family. Here, we unmask the charade and review recent discoveries of UHM-ULM modules for protein-protein interactions. Diverse polypeptide extensions and selective phosphorylation of UHM and ULM family members offer new molecular mechanisms for the assembly of specific partners in the early-stage spliceosome.
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http://dx.doi.org/10.1261/rna.057950.116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5113200PMC
December 2016

Wild-Type U2AF1 Antagonizes the Splicing Program Characteristic of U2AF1-Mutant Tumors and Is Required for Cell Survival.

PLoS Genet 2016 Oct 24;12(10):e1006384. Epub 2016 Oct 24.

Cancer Biology Section, Cancer Genetics Branch, National Human Genome Research Institute, Bethesda, United States Of America.

We have asked how the common S34F mutation in the splicing factor U2AF1 regulates alternative splicing in lung cancer, and why wild-type U2AF1 is retained in cancers with this mutation. A human lung epithelial cell line was genetically modified so that U2AF1S34F is expressed from one of the two endogenous U2AF1 loci. By altering levels of mutant or wild-type U2AF1 in this cell line and by analyzing published data on human lung adenocarcinomas, we show that S34F-associated changes in alternative splicing are proportional to the ratio of S34F:wild-type gene products and not to absolute levels of either the mutant or wild-type factor. Preferential recognition of specific 3' splice sites in S34F-expressing cells is largely explained by differential in vitro RNA-binding affinities of mutant versus wild-type U2AF1 for those same 3' splice sites. Finally, we show that lung adenocarcinoma cell lines bearing U2AF1 mutations do not require the mutant protein for growth in vitro or in vivo. In contrast, wild-type U2AF1 is required for survival, regardless of whether cells carry the U2AF1S34F allele. Our results provide mechanistic explanations of the magnitude of splicing changes observed in U2AF1-mutant cells and why tumors harboring U2AF1 mutations always retain an expressed copy of the wild-type allele.
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http://dx.doi.org/10.1371/journal.pgen.1006384DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5077151PMC
October 2016

An extended U2AF(65)-RNA-binding domain recognizes the 3' splice site signal.

Nat Commun 2016 Mar 8;7:10950. Epub 2016 Mar 8.

Center for RNA Biology and Department of Biochemistry and Biophysics, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642, USA.

How the essential pre-mRNA splicing factor U2AF(65) recognizes the polypyrimidine (Py) signals of the major class of 3' splice sites in human gene transcripts remains incompletely understood. We determined four structures of an extended U2AF(65)-RNA-binding domain bound to Py-tract oligonucleotides at resolutions between 2.0 and 1.5 Å. These structures together with RNA binding and splicing assays reveal unforeseen roles for U2AF(65) inter-domain residues in recognizing a contiguous, nine-nucleotide Py tract. The U2AF(65) linker residues between the dual RNA recognition motifs (RRMs) recognize the central nucleotide, whereas the N- and C-terminal RRM extensions recognize the 3' terminus and third nucleotide. Single-molecule FRET experiments suggest that conformational selection and induced fit of the U2AF(65) RRMs are complementary mechanisms for Py-tract association. Altogether, these results advance the mechanistic understanding of molecular recognition for a major class of splice site signals.
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http://dx.doi.org/10.1038/ncomms10950DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4786784PMC
March 2016

Dividing and Conquering the Family of RNA Recognition Motifs: A Representative Case Based on hnRNP L.

J Mol Biol 2015 Sep 20;427(19):2997-3000. Epub 2015 Jun 20.

Department of Biochemistry and Biophysics, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642, USA. Electronic address:

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http://dx.doi.org/10.1016/j.jmb.2015.06.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4820351PMC
September 2015

Structure-guided U2AF65 variant improves recognition and splicing of a defective pre-mRNA.

Proc Natl Acad Sci U S A 2014 Dec 24;111(49):17420-5. Epub 2014 Nov 24.

Center for RNA Biology and Department of Biochemistry and Biophysics, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642

Purine interruptions of polypyrimidine (Py) tract splice site signals contribute to human genetic diseases. The essential splicing factor U2AF(65) normally recognizes a Py tract consensus sequence preceding the major class of 3' splice sites. We found that neurofibromatosis- or retinitis pigmentosa-causing mutations in the 5' regions of Py tracts severely reduce U2AF(65) affinity. Conversely, we identified a preferred binding site of U2AF(65) for purine substitutions in the 3' regions of Py tracts. Based on a comparison of new U2AF(65) structures bound to either A- or G-containing Py tracts with previously identified pyrimidine-containing structures, we expected to find that a D231V amino acid change in U2AF(65) would specify U over other nucleotides. We found that the crystal structure of the U2AF(65)-D231V variant confirms favorable packing between the engineered valine and a target uracil base. The D231V amino acid change restores U2AF(65) affinity for two mutated splice sites that cause human genetic diseases and successfully promotes splicing of a defective retinitis pigmentosa-causing transcript. We conclude that reduced U2AF(65) binding is a molecular consequence of disease-relevant mutations, and that a structure-guided U2AF(65) variant is capable of manipulating gene expression in eukaryotic cells.
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http://dx.doi.org/10.1073/pnas.1412743111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4267390PMC
December 2014

Cancer-relevant splicing factor CAPERα engages the essential splicing factor SF3b155 in a specific ternary complex.

J Biol Chem 2014 Jun 2;289(25):17325-37. Epub 2014 May 2.

From the Department of Biochemistry and Biophysics, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642 and

U2AF homology motifs (UHMs) mediate protein-protein interactions with U2AF ligand motifs (ULMs) of pre-mRNA splicing factors. The UHM-containing alternative splicing factor CAPERα regulates splicing of tumor-promoting VEGF isoforms, yet the molecular target of the CAPERα UHM is unknown. Here we present structures of the CAPERα UHM bound to a representative SF3b155 ULM at 1.7 Å resolution and, for comparison, in the absence of ligand at 2.2 Å resolution. The prototypical UHM/ULM interactions authenticate CAPERα as a bona fide member of the UHM family of proteins. We identify SF3b155 as the relevant ULM-containing partner of full-length CAPERα in human cell extracts. Isothermal titration calorimetry comparisons of the purified CAPERα UHM binding known ULM-containing proteins demonstrate that high affinity interactions depend on the presence of an intact, intrinsically unstructured SF3b155 domain containing seven ULM-like motifs. The interplay among bound CAPERα molecules gives rise to the appearance of two high affinity sites in the SF3b155 ULM-containing domain. In conjunction with the previously identified, UHM/ULM-mediated complexes of U2AF(65) and SPF45 with SF3b155, this work demonstrates the capacity of SF3b155 to offer a platform for coordinated recruitment of UHM-containing splicing factors.
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http://dx.doi.org/10.1074/jbc.M114.558825DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4067167PMC
June 2014

Staufen1 dimerizes through a conserved motif and a degenerate dsRNA-binding domain to promote mRNA decay.

Nat Struct Mol Biol 2013 Apr 24;20(4):515-24. Epub 2013 Mar 24.

Department of Biochemistry and Biophysics, School of Medicine and Dentistry, University of Rochester, Rochester, New York, USA.

Staufen1 (STAU1)-mediated mRNA decay (SMD) degrades mammalian-cell mRNAs that bind the double-stranded RNA (dsRNA)-binding protein STAU1 in their 3' untranslated region. We report a new motif, which typifies STAU homologs from all vertebrate classes, that is responsible for human STAU1 (hSTAU1) homodimerization. Our crystal structure and mutagenesis analyses reveal that this motif, which we named the Staufen-swapping motif (SSM), and the dsRNA-binding domain 5 ('RBD'5) mediate protein dimerization: the two SSM α-helices of one molecule interact primarily through a hydrophobic patch with the two 'RBD'5 α-helices of a second molecule. 'RBD'5 adopts the canonical α-β-β-β-α fold of a functional RBD, but it lacks residues and features required to bind duplex RNA. In cells, SSM-mediated hSTAU1 dimerization increases the efficiency of SMD by augmenting hSTAU1 binding to the ATP-dependent RNA helicase hUPF1. Dimerization regulates keratinocyte-mediated wound healing and many other cellular processes.
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http://dx.doi.org/10.1038/nsmb.2528DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4096160PMC
April 2013