Publications by authors named "Jermaine L Jenkins"

35 Publications

Affinity and Structural Analysis of the U1A RNA Recognition Motif with Engineered Methionines to Improve Experimental Phasing.

Crystals (Basel) 2021 Mar 10;11(3). Epub 2021 Mar 10.

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

RNA plays a central role in all organisms and can fold into complex structures to orchestrate function. Visualization of such structures often requires crystallization, which can be a bottleneck in the structure-determination process. To promote crystallization, an RNA-recognition motif (RRM) of the U1A spliceosomal protein has been co-opted as a crystallization module. Specifically, the U1-snRNA hairpin II (hpII) single-stranded loop recognized by U1A can be transplanted into an RNA target to promote crystal contacts and to attain phase information via molecular replacement or anomalous diffraction methods using selenomethionine. Herein, we produced the F37M/F77M mutant of U1A to augment the phasing capability of this powerful crystallization module. Selenomethionine-substituted U1A(F37M/F77M) retains high affinity for hpII ( of 59.7 ± 11.4 nM). The 2.20 Å resolution crystal structure reveals that the mutated sidechains make new S-π interactions in the hydrophobic core and are useful for single-wavelength anomalous diffraction. Crystals were also attained of U1A(F37M/F77M) in complex with a bacterial preQ-II riboswitch. The F34M/F37M/F77M mutant was introduced similarly into a lab-evolved U1A variant (TBP6.9) that recognizes the internal bulged loop of HIV-1 TAR RNA. We envision that this short RNA sequence can be placed into non-essential duplex regions to promote crystallization and phasing of target RNAs. We show that selenomethionine-substituted TBP6.9(F34M/F37M/F77M) binds a TAR variant wherein the apical loop was replaced with a GNRA tetraloop ( of 69.8 ± 2.9 nM), laying the groundwork for use of TBP6.9(F34M/F37M/F77M) as a crystallization module. These new tools are available to the research community.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/cryst11030273DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7996396PMC
March 2021

A synthetic small molecule stalls pre-mRNA splicing by promoting an early-stage U2AF2-RNA complex.

Cell Chem Biol 2021 Feb 26. Epub 2021 Feb 26.

Department of Biochemistry and Biophysics and Center for RNA Biology, University of Rochester School of Medicine and Dentistry, Rochester, NY 14620, USA. Electronic address:

Dysregulated pre-mRNA splicing is an emerging Achilles heel of cancers and myelodysplasias. To expand the currently limited portfolio of small-molecule drug leads, we screened for chemical modulators of the U2AF complex, which nucleates spliceosome assembly and is mutated in myelodysplasias. A hit compound specifically enhances RNA binding by a U2AF2 subunit. Remarkably, the compound inhibits splicing of representative substrates and stalls spliceosome assembly at the stage of U2AF function. Computational docking, together with structure-guided mutagenesis, indicates that the compound bridges the tandem U2AF2 RNA recognition motifs via hydrophobic and electrostatic moieties. Cells expressing a cancer-associated U2AF1 mutant are preferentially killed by treatment with the compound. Altogether, our results highlight the potential of trapping early spliceosome assembly as an effective pharmacological means to manipulate pre-mRNA splicing. By extension, we suggest that stabilizing assembly intermediates may offer a useful approach for small-molecule inhibition of macromolecular machines.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.chembiol.2021.02.007DOI Listing
February 2021

An Allosteric Binding Site on Sortilin Regulates the Trafficking of VLDL, PCSK9, and LDLR in Hepatocytes.

Biochemistry 2020 11 5;59(45):4321-4335. Epub 2020 Nov 5.

Department of Biochemistry, University of Illinois Urbana-Champaign, Urbana, Illinois 61801, United States.

ApoB lipoproteins (apo B-Lp) are produced in hepatocytes, and their secretion requires the cargo receptor sortilin. We examined the secretion of apo B-Lp-containing very low-density lipoprotein (VLDL), an LDL progenitor. Sortilin also regulates the trafficking of the subtilase PCSK9, which when secreted binds the LDL receptor (LDLR), resulting in its endocytosis and destruction at the lysosome. We show that the site 2 binding compound (cpd984) has multiple effects in hepatocytes, including (1) enhanced Apo-Lp secretion, (2) increased cellular PCSK9 retention, and (3) augmented levels of LDLR at the plasma membrane. We postulate that cpd984 enhances apo B-Lp secretion in part through binding the lipid phosphatidylinositol 3,4,5-trisphosphate (PIP), which is present at higher levels on circulating VLDL form fed rats relative to after fasting. We attribute the enhanced VLDL secretion to its increased binding affinity for sortilin site 1 induced by cpd984 binding site 2. This hinders PCSK9 binding and secretion, which would subsequently prevent its binding to LDLR leading to its degradation. This suggests that site 2 is an allosteric regulator of site 1 binding. This effect is not limited to VLDL, as cpd984 augments binding of the neuropeptide neurotensin (NT) to sortilin site 1. Molecular dynamics simulations demonstrate that the C-terminus of NT (Ct-NT) stably binds site 1 through an electrostatic interaction. This was bolstered by the ability of Ct-NT to disrupt lower-affinity interactions between sortilin and the site 1 ligand PIP. Together, these data show that binding cargo at sortilin site 1 is allosterically regulated through site 2 binding, with important ramifications for cellular lipid homeostasis involving proteins such as PCSK9 and LDLR.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acs.biochem.0c00741DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7674269PMC
November 2020

Co-crystal structures of HIV TAR RNA bound to lab-evolved proteins show key roles for arginine relevant to the design of cyclic peptide TAR inhibitors.

J Biol Chem 2020 12 13;295(49):16470-16486. Epub 2020 Oct 13.

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

RNA-protein interfaces control key replication events during the HIV-1 life cycle. The viral -activator of transcription (Tat) protein uses an archetypal arginine-rich motif (ARM) to recruit the host positive transcription elongation factor b (pTEFb) complex onto the viral -activation response (TAR) RNA, leading to activation of HIV transcription. Efforts to block this interaction have stimulated production of biologics designed to disrupt this essential RNA-protein interface. Here, we present four co-crystal structures of lab-evolved TAR-binding proteins (TBPs) in complex with HIV-1 TAR. Our results reveal that high-affinity binding requires a distinct sequence and spacing of arginines within a specific β2-β3 hairpin loop that arose during selection. Although loops with as many as five arginines were analyzed, only three arginines could bind simultaneously with major-groove guanines. Amino acids that promote backbone interactions within the β2-β3 loop were also observed to be important for high-affinity interactions. Based on structural and affinity analyses, we designed two cyclic peptide mimics of the TAR-binding β2-β3 loop sequences present in two high-affinity TBPs ( values of 4.2 ± 0.3 and 3.0 ± 0.3 nm). Our efforts yielded low-molecular weight compounds that bind TAR with low micromolar affinity ( values ranging from 3.6 to 22 μm). Significantly, one cyclic compound within this series blocked binding of the Tat-ARM peptide to TAR in solution assays, whereas its linear counterpart did not. Overall, this work provides insight into protein-mediated TAR recognition and lays the ground for the development of cyclic peptide inhibitors of a vital HIV-1 RNA-protein interaction.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.RA120.015444DOI Listing
December 2020

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

J Biol Chem 2020 12 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. Electronic address:

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.RA120.015339DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7863893PMC
December 2020

Analysis of a preQ1-I riboswitch in effector-free and bound states reveals a metabolite-programmed nucleobase-stacking spine that controls gene regulation.

Nucleic Acids Res 2020 08;48(14):8146-8164

Department of Biochemistry & Biophysics, University of Rochester School of Medicine & Dentistry, Rochester, NY 14642, USA.

Riboswitches are structured RNA motifs that recognize metabolites to alter the conformations of downstream sequences, leading to gene regulation. To investigate this molecular framework, we determined crystal structures of a preQ1-I riboswitch in effector-free and bound states at 2.00 Å and 2.65 Å-resolution. Both pseudoknots exhibited the elusive L2 loop, which displayed distinct conformations. Conversely, the Shine-Dalgarno sequence (SDS) in the S2 helix of each structure remained unbroken. The expectation that the effector-free state should expose the SDS prompted us to conduct solution experiments to delineate environmental changes to specific nucleobases in response to preQ1. We then used nudged elastic band computational methods to derive conformational-change pathways linking the crystallographically-determined effector-free and bound-state structures. Pathways featured: (i) unstacking and unpairing of L2 and S2 nucleobases without preQ1-exposing the SDS for translation and (ii) stacking and pairing L2 and S2 nucleobases with preQ1-sequestering the SDS. Our results reveal how preQ1 binding reorganizes L2 into a nucleobase-stacking spine that sequesters the SDS, linking effector recognition to biological function. The generality of stacking spines as conduits for effector-dependent, interdomain communication is discussed in light of their existence in adenine riboswitches, as well as the turnip yellow mosaic virus ribosome sensor.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/nar/gkaa546DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7641330PMC
August 2020

A small-molecule competitive inhibitor of phosphatidic acid binding by the AAA+ protein NSF/Sec18 blocks the SNARE-priming stage of vacuole fusion.

J Biol Chem 2019 11 12;294(46):17168-17185. Epub 2019 Sep 12.

Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801

The homeostasis of most organelles requires membrane fusion mediated by oluble -ethylmaleimide-sensitive factor (NSF) ttachment protein ceptors (SNAREs). SNAREs undergo cycles of activation and deactivation as membranes move through the fusion cycle. At the top of the cycle, inactive -SNARE complexes on a single membrane are activated, or primed, by the hexameric ATPase associated with the diverse cellular activities (AAA+) protein, -ethylmaleimide-sensitive factor (NSF/Sec18), and its co-chaperone α-SNAP/Sec17. Sec18-mediated ATP hydrolysis drives the mechanical disassembly of SNAREs into individual coils, permitting a new cycle of fusion. Previously, we found that Sec18 monomers are sequestered away from SNAREs by binding phosphatidic acid (PA). Sec18 is released from the membrane when PA is hydrolyzed to diacylglycerol by the PA phosphatase Pah1. Although PA can inhibit SNARE priming, it binds other proteins and thus cannot be used as a specific tool to further probe Sec18 activity. Here, we report the discovery of a small-molecule compound, we call IPA (inhibitor of priming activity), that binds Sec18 with high affinity and blocks SNARE activation. We observed that IPA blocks SNARE priming and competes for PA binding to Sec18. Molecular dynamics simulations revealed that IPA induces a more rigid NSF/Sec18 conformation, which potentially disables the flexibility required for Sec18 to bind to PA or to activate SNAREs. We also show that IPA more potently and specifically inhibits NSF/Sec18 activity than does ethylmaleimide, requiring the administration of only low micromolar concentrations of IPA, demonstrating that this compound could help to further elucidate SNARE-priming dynamics.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.RA119.008865DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6873166PMC
November 2019

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1261/rna.070649.119DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6633205PMC
August 2019

Platelet-derived β2M regulates monocyte inflammatory responses.

JCI Insight 2019 03 7;4(5). Epub 2019 Mar 7.

Aab Cardiovascular Research Institute.

β-2 Microglobulin (β2M) is a molecular chaperone for the major histocompatibility class I (MHC I) complex, hemochromatosis factor protein (HFE), and the neonatal Fc receptor (FcRn), but β2M may also have less understood chaperone-independent functions. Elevated plasma β2M has a direct role in neurocognitive decline and is a risk factor for adverse cardiovascular events. β2M mRNA is present in platelets at very high levels, and β2M is part of the activated platelet releasate. In addition to their more well-studied thrombotic functions, platelets are important immune regulatory cells that release inflammatory molecules and contribute to leukocyte trafficking, activation, and differentiation. We have now found that platelet-derived β2M is a mediator of monocyte proinflammatory differentiation through noncanonical TGFβ receptor signaling. Circulating monocytes from mice lacking β2M only in platelets (Plt-β2M-/-) had a more proreparative monocyte phenotype, in part dependent on increased platelet-derived TGFβ signaling in the absence of β2M. Using a mouse myocardial infarction (MI) model, Plt-β2M-/- mice had limited post-MI proinflammatory monocyte responses and, instead, demonstrated early proreparative monocyte differentiation, profibrotic myofibroblast responses, and a rapid decline in heart function compared with WT mice. These data demonstrate a potentially novel chaperone-independent, monocyte phenotype-regulatory function for platelet β2M and that platelet-derived 2M and TGFβ have opposing roles in monocyte differentiation that may be important in tissue injury responses.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1172/jci.insight.122943DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6483513PMC
March 2019

Phosphatidic acid induces conformational changes in Sec18 protomers that prevent SNARE priming.

J Biol Chem 2019 03 7;294(9):3100-3116. Epub 2019 Jan 7.

From the Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801,

Eukaryotic cell homeostasis requires transfer of cellular components among organelles and relies on membrane fusion catalyzed by SNARE proteins. Inactive SNARE bundles are reactivated by hexameric -ethylmaleimide-sensitive factor, vesicle-fusing ATPase (Sec18/NSF)-driven disassembly that enables a new round of membrane fusion. We previously found that phosphatidic acid (PA) binds Sec18 and thereby sequesters it from SNAREs and that PA dephosphorylation dissociates Sec18 from the membrane, allowing it to engage SNARE complexes. We now report that PA also induces conformational changes in Sec18 protomers and that hexameric Sec18 cannot bind PA membranes. Molecular dynamics (MD) analyses revealed that the D1 and D2 domains of Sec18 contain PA-binding sites and that the residues needed for PA binding are masked in hexameric Sec18. Importantly, these simulations also disclosed that a major conformational change occurs in the linker region between the D1 and D2 domains, which is distinct from the conformational changes that occur in hexameric Sec18 during SNARE priming. Together, these findings indicate that PA regulates Sec18 function by altering its architecture and stabilizing membrane-bound Sec18 protomers.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.RA118.006552DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6398130PMC
March 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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.RA118.006764DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6393619PMC
February 2019

Use of Surface Plasmon Resonance (SPR) to Determine Binding Affinities and Kinetic Parameters Between Components Important in Fusion Machinery.

Methods Mol Biol 2019 ;1860:199-210

Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, IL, USA.

Surface plasmon resonance (SPR) can be used to analyze both binding affinities and kinetic parameters between a ligand and an analyte. SPR can be performed by either cross-linking a given ligand to a sensor chip covalently or utilizing high-affinity non-covalent interactions to secure a ligand in a particular conformation to a chip, both of which have their potential advantages. SPR measurements are mass based and reflect the proportional amount of analyte bound to a given ligand at a given concentration when flowed at a set rate in order to determine the binding parameters of a given biochemical interaction. The resultant sensorgram can indicate different types of binding events as well as provide both k and k which can be used to determine an equilibrium dissociation constant K. SPR can be used to measure binding affinity of proteins involved in fusion such as between SNAREs, SNAREs, and proteins that interact with them such as Sec18 (NSF) or Sec17 (alpha-SNAP), or to measure the binding of any fusion-related protein to a specific lipid or other small molecules; however, Ks are determined by SPR using a titration of concentrations of analyte and a maximum point on the sensorgram signifying saturation of the protein in order to determine a steady-state K.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/978-1-4939-8760-3_12DOI Listing
June 2019

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acs.biochem.7b00551DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6005654PMC
September 2017

Metalloriboswitches: RNA-based inorganic ion sensors that regulate genes.

J Biol Chem 2017 06 28;292(23):9441-9450. Epub 2017 Apr 28.

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

Divalent ions fulfill essential cellular roles and are required for virulence by certain bacteria. Free intracellular Mg can approach 5 mm, but at this level Mn, Ni, or Co can be growth-inhibitory, and magnesium fluoride is toxic. To maintain ion homeostasis, many bacteria have evolved ion sensors embedded in the 5'-leader sequences of mRNAs encoding ion uptake or efflux channels. Here, we review current insights into these "metalloriboswitches," emphasizing ion-specific binding by structured RNA aptamers and associated conformational changes in downstream signal sequences. This riboswitch-effector interplay produces a layer of gene regulatory feedback that has elicited interest as an antibacterial target.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.R117.787713DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5465474PMC
June 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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.tig.2017.03.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5447463PMC
May 2017

Human CaaX protease ZMPSTE24 expressed in yeast: Structure and inhibition by HIV protease inhibitors.

Protein Sci 2017 02 11;26(2):242-257. Epub 2016 Nov 11.

Department of Pediatrics, University of Rochester Medical Center, Rochester, New York, 14642.

The function and localization of proteins and peptides containing C-terminal "CaaX" (Cys-aliphatic-aliphatic-anything) sequence motifs are modulated by post-translational attachment of isoprenyl groups to the cysteine sulfhydryl, followed by proteolytic cleavage of the aaX amino acids. The zinc metalloprotease ZMPSTE24 is one of two enzymes known to catalyze this cleavage. The only identified target of mammalian ZMPSTE24 is prelamin A, the precursor to the nuclear scaffold protein lamin A. ZMPSTE24 also cleaves prelamin A at a second site 15 residues upstream from the CaaX site. Mutations in ZMPSTE24 result in premature-aging diseases and inhibition of ZMPSTE24 activity has been reported to be an off-target effect of HIV protease inhibitors. We report here the expression (in yeast), purification, and crystallization of human ZMPSTE24 allowing determination of the structure to 2.0 Å resolution. Compared to previous lower resolution structures, the enhanced resolution provides: (1) a detailed view of the active site of ZMPSTE24, including water coordinating the catalytic zinc; (2) enhanced visualization of fenestrations providing access from the exterior to the interior cavity of the protein; (3) a view of the C-terminus extending away from the main body of the protein; (4) localization of ordered lipid and detergent molecules at internal and external surfaces and also projecting through fenestrations; (5) identification of water molecules associated with the surface of the internal cavity. We also used a fluorogenic assay of the activity of purified ZMPSTE24 to demonstrate that HIV protease inhibitors directly inhibit the human enzyme in a manner indicative of a competitive mechanism.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/pro.3074DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5275749PMC
February 2017

Phosphatidylinositol (3,4,5)-trisphosphate binds to sortilin and competes with neurotensin: Implications for very low density lipoprotein binding.

Biochem Biophys Res Commun 2016 Oct 22;479(3):551-556. Epub 2016 Sep 22.

Department of Pathology and Laboratory Medicine, University of Rochester Medical Center, Rochester, NY 14642, USA. Electronic address:

Sortilin is a multi-ligand sorting receptor that interacts with B100-containing VLDL and LDL as well as other ligands including neurotensin (NT). The current study investigates the hypothesis that phosphatidylinositol (3,4,5)-trisphosphate (PIP3) generated downstream of insulin action can directly bind to sortilin. NT binds to sortilin at a well characterized site via its carboxy terminus (C-term). Using a crystal structure of human sortilin (hsortilin), PIP3 is predicted to bind at this C-term site. Binding of PIP3 to hsortilin is demonstrated using surface plasmon resonance (SPR) flowing PIP3 nanodiscs over immobilized hsortilin. Studies were performed using SPR where dibutanoyl PIP3 is shown to compete with NT for sortilin binding. Rat VLDL and LDL were evaluated for PIP3 content immunologically using monoclonal antibodies directed against PIP3. Rat plasma VLDL contained three times more immunoreactive PIP3 than LDL per μg of protein. Because VLDL contains additional ligands that bind sortilin, to distinguish specific PIP3 binding, we used PIP3 liposomes. Liposome floatation assays were used to demonstrate PIP3 liposome binding to sortilin. Using SPR and immobilized hsortilin, the C-term NT tetrapeptide (P-Y-I-L) is shown to bind to hsortilin. A compound (cpd984) was identified with strong theoretical binding to the site on sortilin involved in NT N-terminal binding. When cpd984 is co-incubated with the tetrapeptide, the affinity of binding to sortilin is increased. Similarly, the affinity of PIP3 liposome binding increased in the presence of cpd984. Overall, results demonstrate that sortilin is a PIP3 binding protein with binding likely to occur at the C-term NT binding site. The presence of multiple ligands on B100-containing lipoproteins, VLDL and LDL, raises the interesting possibility for increased interaction with sortilin based on the presence of PIP3.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5056161PMC
http://dx.doi.org/10.1016/j.bbrc.2016.09.108DOI Listing
October 2016

The Quick and the Dead: A Guide to Fast Phasing of Small Ribozyme and Riboswitch Crystal Structures.

Methods Mol Biol 2016 ;1490:265-80

Department of Biochemistry and Biophysics, University of Rochester School of Medicine and Dentistry, 601 Elmwood Avenue, Box 712, Rochester, NY, 14642, USA.

Ribozymes and riboswitches are examples of non-protein-coding (nc)RNA molecules that achieve biological activity by adopting complex three-dimensional folds. Visualization of such molecules at near-atomic resolution can enhance our understanding of how chemical groups are organized spatially, thereby providing novel insight into function. This approach has its challenges, which mainly entail sample crystallization followed by the application of empirical, structure-determination methods that often include experimental "phasing" of X-ray diffraction data. A paucity of high-quality crystals or a low symmetry space group are factors that demand rapid assessment of phasing potential during an ongoing experiment in order to assure a successful outcome. Here we describe the process of evaluating the anomalous signal-to-noise as a prelude to single wavelength or multiwavelength anomalous diffraction (SAD or MAD) phasing. Test cases include an autolytic 62-mer RNA enzyme known as the hairpin ribozyme, and a 33-mer riboswitch that binds the modified guanine metabolite preQ1. The crystals were derivatized with iridium (III) hexammine and osmium (III) pentaammine triflate, respectively. Each data set was then subjected to the XPREP and SHELX programs to assess the anomalous signal-to-noise and to locate the heavy-atom substructure. Subsequent noise filtering was conducted in SHELXE or RESOLVE. The methods described are applicable to the rapid phasing of RNA X-ray diffraction data, and contrast the efficacy of in-house X-rays with those attainable from synchrotron-radiation sources in terms of the potential to plan for and execute an experimental structure determination.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/978-1-4939-6433-8_17DOI Listing
January 2018

Sortilin facilitates VLDL-B100 secretion by insulin sensitive McArdle RH7777 cells.

Biochem Biophys Res Commun 2016 09 2;478(2):546-52. Epub 2016 Aug 2.

Department of Pathology and Laboratory Medicine, University of Rochester Medical Center, Rochester, NY 14642, USA. Electronic address:

Studies examining the relationship between cellular sortilin and VLDL-B100 secretion demonstrate inconsistent results. Current studies explore the possibility that discrepancies may be related to insulin sensitivity. McArdle RH7777 cells (McA cells) cultured under serum enriched conditions lose sensitivity to insulin. Following incubation in serum-free DMEM containing 1% BSA, McA cells become insulin responsive and demonstrate reduced apo B secretion. Current studies indicate that insulin sensitive McA cells express lower cellular sortilin that corresponds with reduction in VLDL-B100 secretion without changes in mRNA of either sortilin or apo B. When sortilin expression is further reduced by siRNA knockdown (KD), there are additional decreases in VLDL-B100 secretion. A crystal structure of human sortilin (hsortilin) identifies two binding sites on the luminal domain for the N- and C-termini of neurotensin (NT). A small organic compound (cpd984) was identified that has strong theoretical binding to the N-terminal site. Both cpd984 and NT bind hsortilin by surface plasmon resonance. In incubations with insulin sensitive McA cells, cpd984 was shown to enhance VLDL-B100 secretion at each level of sortilin KD suggesting cpd984 acted through sortilin in mediating its effect. Current results support a role for sortilin to facilitate VLDL-B100 secretion which is limited to insulin sensitive McA cells. Inconsistent reports of the relationship between VLDL-B100 secretion and sortilin in previous studies may relate to differing functions of sortilin in VLDL-B100 secretion depending upon insulin sensitivity.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bbrc.2016.07.096DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5002383PMC
September 2016

Interaction of human biliverdin reductase with Akt/protein kinase B and phosphatidylinositol-dependent kinase 1 regulates glycogen synthase kinase 3 activity: a novel mechanism of Akt activation.

FASEB J 2016 08 10;30(8):2926-44. Epub 2016 May 10.

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

Biliverdin reductase A (BVR) and Akt isozymes have overlapping pleiotropic functions in the insulin/PI3K/MAPK pathway. Human BVR (hBVR) also reduces the hemeoxygenase activity product biliverdin to bilirubin and is directly activated by insulin receptor kinase (IRK). Akt isoenzymes (Akt1-3) are downstream of IRK and are activated by phosphatidylinositol-dependent kinase 1 (PDK1) phosphorylating T(308) before S(473) autophosphorylation. Akt (RxRxxSF) and PDK1 (RFxFPxFS) binding motifs are present in hBVR. Phosphorylation of glycogen synthase kinase 3 (GSK3) isoforms α/β by Akts inhibits their activity; nonphosphorylated GSK3β inhibits activation of various genes. We examined the role of hBVR in PDK1/Akt1/GSK3 signaling and Akt1 in hBVR phosphorylation. hBVR activates phosphorylation of Akt1 at S(473) independent of hBVR's kinase competency. hBVR and Akt1 coimmunoprecipitated, and in-cell Förster resonance energy transfer (FRET) and glutathione S-transferase pulldown analyses identified Akt1 pleckstrin homology domain as the interactive domain. hBVR activates phosphorylation of Akt1 at S(473) independent of hBVR's kinase competency. Site-directed mutagenesis, mass spectrometry, and kinetic analyses identified S(230) in hBVR (225)RNRYLSF sequence as the Akt1 target. Underlined amino acids are the essential residues of the signaling motifs. In cells, hBVR-activated Akt1 increased both GSK3α/β and forkhead box of the O class transcription class 3 (FoxO3) phosphorylation and inhibited total GSK3 activity; depletion of hBVR released inhibition and stimulated glucose uptake. Immunoprecipitation analysis showed that PDK1 and hBVR interact through hBVR's PDK1 binding (161)RFGFPAFS motif and formation of the PDK1/hBVR/Akt1 complex. sihBVR blocked complex formation. Findings identify hBVR as a previously unknown coactivator of Akt1 and as a key mediator of Akt1/GSK3 pathway, as well as define a key role for hBVR in Akt1 activation by PDK1.-Miralem, T., Lerner-Marmarosh, N., Gibbs, P. E. M., Jenkins, J. L., Heimiller, C., Maines, M. D. Interaction of human biliverdin reductase with Akt/protein kinase B and phosphatidylinositol-dependent kinase 1 regulates glycogen synthase kinase 3 activity: a novel mechanism of Akt activation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1096/fj.201600330RRDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4970609PMC
August 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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/ncomms10950DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4786784PMC
March 2016

ITC analysis of ligand binding to preQ₁ riboswitches.

Methods Enzymol 2014 ;549:435-50

Department of Biochemistry & Biophysics, University of Rochester School of Medicine and Dentistry, Rochester, New York, USA; Center for RNA Biology, University of Rochester School of Medicine and Dentistry, Rochester, New York, USA; Structural Biology & Biophysics Facility, University of Rochester School of Medicine and Dentistry, Rochester, New York, USA. Electronic address:

Riboswitches regulate genes by binding to small-molecule effectors. Isothermal titration calorimetry (ITC) provides a label-free method to quantify the equilibrium association constant, K(A), of a riboswitch interaction with its cognate ligand. In addition to probing affinity and specific chemical contributions that contribute to binding, ITC can be used to measure the thermodynamic parameters of an interaction (ΔG, ΔH, and ΔS), in addition to the binding stoichiometry (N). Here, we describe methods developed to measure the binding affinity of various preQ1 riboswitch classes for the pyrrolopyrimidine effector, preQ1. Example isotherms are provided along with a review of various preQ1-II (class 2) riboswitch mutants that were interrogated by ITC to quantify the energetic contributions of specific interactions visualized in the crystal structure. Protocols for ITC are provided in sufficient detail that the reader can reproduce experiments independently, or develop derivative methods suitable for analyzing novel riboswitch-ligand binding interactions.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/B978-0-12-801122-5.00018-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5749633PMC
July 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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.1412743111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4267390PMC
December 2014

U2AF65 adapts to diverse pre-mRNA splice sites through conformational selection of specific and promiscuous RNA recognition motifs.

Nucleic Acids Res 2013 Apr 1;41(6):3859-73. Epub 2013 Feb 1.

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

Degenerate splice site sequences mark the intron boundaries of pre-mRNA transcripts in multicellular eukaryotes. The essential pre-mRNA splicing factor U2AF(65) is faced with the paradoxical tasks of accurately targeting polypyrimidine (Py) tracts preceding 3' splice sites while adapting to both cytidine and uridine nucleotides with nearly equivalent frequencies. To understand how U2AF(65) recognizes degenerate Py tracts, we determined six crystal structures of human U2AF(65) bound to cytidine-containing Py tracts. As deoxy-ribose backbones were required for co-crystallization with these Py tracts, we also determined two baseline structures of U2AF(65) bound to the deoxy-uridine counterparts and compared the original, RNA-bound structure. Local structural changes suggest that the N-terminal RNA recognition motif 1 (RRM1) is more promiscuous for cytosine-containing Py tracts than the C-terminal RRM2. These structural differences between the RRMs were reinforced by the specificities of wild-type and site-directed mutant U2AF(65) for region-dependent cytosine- and uracil-containing RNA sites. Small-angle X-ray scattering analyses further demonstrated that Py tract variations select distinct inter-RRM spacings from a pre-existing ensemble of U2AF(65) conformations. Our results highlight both local and global conformational selection as a means for universal 3' splice site recognition by U2AF(65).
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/nar/gkt046DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3616741PMC
April 2013

A transition-state interaction shifts nucleobase ionization toward neutrality to facilitate small ribozyme catalysis.

J Am Chem Soc 2012 Oct 3;134(41):16933-6. Epub 2012 Oct 3.

Department of Biochemistry and Biophysics, Center for RNA Biology, University of Rochester School of Medicine & Dentistry, 601 Elmwood Avenue, Box 712, Rochester, New York 14642, USA.

One mechanism by which ribozymes can accelerate biological reactions is by adopting folds that favorably perturb nucleobase ionization. Herein we used Raman crystallography to directly measure pK(a) values for the Ade38 N1 imino group of a hairpin ribozyme in distinct conformational states. A transition-state analogue gave a pK(a) value of 6.27 ± 0.05, which agrees strikingly well with values measured by pH-rate analyses. To identify the chemical attributes that contribute to the shifted pK(a), we determined crystal structures of hairpin ribozyme variants containing single-atom substitutions at the active site and measured their respective Ade38 N1 pK(a) values. This approach led to the identification of a single interaction in the transition-state conformation that elevates the base pK(a) > 0.8 log unit relative to the precatalytic state. The agreement of the microscopic and macroscopic pK(a) values and the accompanying structural analysis supports a mechanism in which Ade38 N1(H)+ functions as a general acid in phosphodiester bond cleavage. Overall the results quantify the contribution of a single electrostatic interaction to base ionization, which has broad relevance for understanding how RNA structure can control chemical reactivity.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/ja3070528DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3598154PMC
October 2012

A Broad range of conformations contribute to the solution ensemble of the essential splicing factor U2AF(65).

Biochemistry 2012 Jul 19;51(26):5223-5. Epub 2012 Jun 19.

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

U2AF(65) is essential for pre-mRNA splicing in most eukaryotes. Two consecutive RNA recognition motifs (RRM) of U2AF(65) recognize a polypyrimidine tract at the 3' splice site. Here, we use small-angle X-ray scattering to demonstrate that the tandem U2AF(65) RRMs exhibit a broad range of conformations in the solution ensemble. The majority of U2AF(65) conformations exhibit few contacts between the RRMs, such as observed in the crystal structure. A subpopulation adopts tight inter-RRM contacts, such as independently reported based on paramagnetic relaxation enhancements. These complementary structural methods demonstrate that diverse splice sites have the opportunity to select compact or extended inter-RRM proximities from the U2AF(65) conformational pool.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/bi300277tDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3447987PMC
July 2012

Crystallographic analysis of small ribozymes and riboswitches.

Methods Mol Biol 2012 ;848:159-84

Department of Biochemistry and Biophysics, University of Rochester, Rochester, NY, USA.

Ribozymes and riboswitches are RNA motifs that accelerate biological reactions and regulate gene expression in response to metabolite recognition, respectively. These RNA molecules gain functionality via complex folding that cannot be predicted a priori, and thus requires high-resolution three-dimensional structure determination to locate key functional attributes. Herein, we present an overview of the methods used to determine small RNA structures with an emphasis on RNA preparation, crystallization, and structure refinement. We draw upon examples from our own research in the analysis of the leadzyme ribozyme, the hairpin ribozyme, a class I preQ(1) riboswitch, and variants of a larger class II preQ(1) riboswitch. The methods presented provide a guide for comparable investigations of noncoding RNA molecules including a 48-solution, "first choice" RNA crystal screen compiled from our prior successes with commercially available screens.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/978-1-61779-545-9_11DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5008910PMC
May 2012

Three RNA recognition motifs participate in RNA recognition and structural organization by the pro-apoptotic factor TIA-1.

J Mol Biol 2012 Jan 2;415(4):727-40. Epub 2011 Dec 2.

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

T-cell intracellular antigen-1 (TIA-1) regulates developmental and stress-responsive pathways through distinct activities at the levels of alternative pre-mRNA splicing and mRNA translation. The TIA-1 polypeptide contains three RNA recognition motifs (RRMs). The central RRM2 and C-terminal RRM3 associate with cellular mRNAs. The N-terminal RRM1 enhances interactions of a C-terminal Q-rich domain of TIA-1 with the U1-C splicing factor, despite linear separation of the domains in the TIA-1 sequence. Given the expanded functional repertoire of the RRM family, it was unknown whether TIA-1 RRM1 contributes to RNA binding as well as documented protein interactions. To address this question, we used isothermal titration calorimetry and small-angle X-ray scattering to dissect the roles of the TIA-1 RRMs in RNA recognition. Notably, the fas RNA exhibited two binding sites with indistinguishable affinities for TIA-1. Analyses of TIA-1 variants established that RRM1 was dispensable for binding AU-rich fas sites, yet all three RRMs were required to bind a polyU RNA with high affinity. Small-angle X-ray scattering analyses demonstrated a "V" shape for a TIA-1 construct comprising the three RRMs and revealed that its dimensions became more compact in the RNA-bound state. The sequence-selective involvement of TIA-1 RRM1 in RNA recognition suggests a possible role for RNA sequences in regulating the distinct functions of TIA-1. Further implications for U1-C recruitment by the adjacent TIA-1 binding sites of the fas pre-mRNA and the bent TIA-1 shape, which organizes the N- and C-termini on the same side of the protein, are discussed.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jmb.2011.11.040DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3282181PMC
January 2012

Comparison of a preQ1 riboswitch aptamer in metabolite-bound and free states with implications for gene regulation.

J Biol Chem 2011 Jul 18;286(28):24626-37. Epub 2011 May 18.

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

Riboswitches are RNA regulatory elements that govern gene expression by recognition of small molecule ligands via a high affinity aptamer domain. Molecular recognition can lead to active or attenuated gene expression states by controlling accessibility to mRNA signals necessary for transcription or translation. Key areas of inquiry focus on how an aptamer attains specificity for its effector, the extent to which the aptamer folds prior to encountering its ligand, and how ligand binding alters expression signal accessibility. Here we present crystal structures of the preQ(1) riboswitch from Thermoanaerobacter tengcongensis in the preQ(1)-bound and free states. Although the mode of preQ(1) recognition is similar to that observed for preQ(0), surface plasmon resonance revealed an apparent K(D) of 2.1 ± 0.3 nm for preQ(1) but a value of 35.1 ± 6.1 nm for preQ(0). This difference can be accounted for by interactions between the preQ(1) methylamine and base G5 of the aptamer. To explore conformational states in the absence of metabolite, the free-state aptamer structure was determined. A14 from the ceiling of the ligand pocket shifts into the preQ(1)-binding site, resulting in "closed" access to the metabolite while simultaneously increasing exposure of the ribosome-binding site. Solution scattering data suggest that the free-state aptamer is compact, but the "closed" free-state crystal structure is inadequate to describe the solution scattering data. These observations are distinct from transcriptional preQ(1) riboswitches of the same class that exhibit strictly ligand-dependent folding. Implications for gene regulation are discussed.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.M111.230375DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3137038PMC
July 2011

Large favorable enthalpy changes drive specific RNA recognition by RNA recognition motif proteins.

Biochemistry 2011 Mar 2;50(9):1429-31. Epub 2011 Feb 2.

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

The RNA recognition motif (RRM) is a prevalent class of RNA binding domains. Although a number of RRM/RNA structures have been determined, thermodynamic analyses are relatively uncommon. Here, we use isothermal titration calorimetry to characterize single-stranded (ss)RNA binding by four representative RRM-containing proteins: (i) U2AF(65), (ii) SXL, (iii) TIA-1, and (iv) PAB. In all cases, ssRNA binding is accompanied by remarkably large favorable enthalpy changes (-30 to -60 kcal mol(-1)) and unfavorable entropy changes. Alterations of key RRM residues and binding sites indicate that under the nearly physiological conditions of these studies, large thermodynamic changes represent a signature of specific ssRNA recognition by RRMs.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/bi102057mDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3050080PMC
March 2011