Mohammad Salim

Structure of a class II preQ1 riboswitch reveals ligand recognition by a new fold

PreQ1 riboswitches regulate genes by binding the pyrrolopyrimidine intermediate preQ1 during biosynthesis of the essential tRNA base queuosine. We report the first preQ1-II riboswitch structure at 2.3 Å resolution, which uses a novel fold to achieve effector recognition at the confluence of a three-way-helical junction flanking a pseudoknotted ribosome-binding site (RBS). The results account for preQ1-II-riboswitch-mediated translational control, and expand the known repertoire of ligand binding modes utilized by regulatory RNAs.

The postmortem Alzheimer brain is a source of structurally and functionally intact astrocytic messenger RNA

Although the precise role of astrocytes in the pathogenesis of Alzheimers disease (AD) is currently undefined, studies carried out at the molecular level may lead to new insights into the functioning of this class of brain cells in dementia. In order to facilitate such investigations, methods are described that establish that structurally and functionally intact messenger RNA (mRNA) for an astrocytic marker, glial fibrillary acidic protein (GFAP), is present in the postmortem Alzheimers disease brain after long postmortem intervals. Rapid preparative procedures were used to obtain poly(A)+ RNA from postmortem control and AD cortices. In vitro protein synthesis was carried out in a reticulocyte system. Relative to controls, AD mRNA synthesized a two-fold higher level of a 50,000 mol.wt. protein that was immunologically identified as GFAP. High levels of GFAP synthesis by purified mRNA from AD cortices was independent of age at death and postmortem interval up to 24 h. Northern blot hybridization using a cloned human GFAP riboprobe was used to evaluate postmortem GFAP mRNA stability. No appreciable degradation products of GFAP mRNA were observed on Northern blots for at least 10 h postmortem in poly(A)+ RNA extracted from the AD brain. The described methodology demonstrates that the postmortem AD brain is an excellent source of functionally and structurally intact astrocyte-specific mRNA.

NMDA and AMPA receptors in transgenic mice expressing human β-amyloid protein

The human β‐amyloid protein may play an important, possibly primary, role in the pathogenesis of Alzheimers disease (AD), and it appears to potentiate the susceptibility of neurons to excitotoxicity. AD is associated with alterations in the W‐methyl‐D‐aspartate (NMDA) and a‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐proprionic acid (AMPA) subtypes of glutamate receptors, and it has been suggested that excitotoxicity may play a role in neuronal damage in AD. In this study, we have used quantitative receptor autoradiography to examine NMDA and AMPA receptors in transgenic mice that contain the gene for the carboxyl‐terminal 100 amino acids of the human amyloid precursor protein, beginning with the β‐amyloid region, which is under the control of the JC viral early region promoter. Reverse transcriptase‐polymerase chain reaction confirmed that the brains of transgenic mice expressed β‐amyloid mRNA and that control mice did not. NMDA receptors, assessed with [3H]MK‐801, were unchanged in the transgenic compared with the control mice. In the transgenic mice, there were no significant changes in [3H]AMPA receptor binding compared with controls. This study represents the first attempt to evaluate in transgenic mice the in vivo interaction between β‐amyloid expression and excitatory amino acid receptors.

Structural analysis of a class III preQ1 riboswitch reveals an aptamer distant from a ribosome-binding site regulated by fast dynamics

Significance Riboswitches are RNA molecules found mostly in bacteria that control genes by sensing cellular levels of metabolites, such as the simple organic compound preQ1. The diversity of riboswitches and their potential as novel antibiotic targets continue to elicit interest in these regulatory sequences. Here we present the crystal structure of a newly discovered bacterial preQ1-III riboswitch that senses preQ1 using an unusual, two-part architecture. A complementary analysis of flexibility and dynamics showed that recognition of preQ1 induces riboswitch compaction, while concomitantly enhancing formation of a distant double-helix possessing a regulatory signal that zips and unzips rapidly, producing gene “off” and “on” states. These observations expand our knowledge of riboswitch construction and suggest a broader role for dynamics than previously recognized. PreQ1-III riboswitches are newly identified RNA elements that control bacterial genes in response to preQ1 (7-aminomethyl-7-deazaguanine), a precursor to the essential hypermodified tRNA base queuosine. Although numerous riboswitches fold as H-type or HLout-type pseudoknots that integrate ligand-binding and regulatory sequences within a single folded domain, the preQ1-III riboswitch aptamer forms a HLout-type pseudoknot that does not appear to incorporate its ribosome-binding site (RBS). To understand how this unusual organization confers function, we determined the crystal structure of the class III preQ1 riboswitch from Faecalibacterium prausnitzii at 2.75 Å resolution. PreQ1 binds tightly (KD,app 6.5 ± 0.5 nM) between helices P1 and P2 of a three-way helical junction wherein the third helix, P4, projects orthogonally from the ligand-binding pocket, exposing its stem-loop to base pair with the 3′ RBS. Biochemical analysis, computational modeling, and single-molecule FRET imaging demonstrated that preQ1 enhances P4 reorientation toward P1–P2, promoting a partially nested, H-type pseudoknot in which the RBS undergoes rapid docking (kdock ∼0.6 s−1) and undocking (kundock ∼1.1 s−1). Discovery of such dynamic conformational switching provides insight into how a riboswitch with bipartite architecture uses dynamics to modulate expression platform accessibility, thus expanding the known repertoire of gene control strategies used by regulatory RNAs.

Preparation of a recombinant cDNA library from poly(A+) RNA of the Alzheimer brain. Identification and characterization of a cDNA copy encoding a glial-specific protein.

Studies were undertaken to assess the extent to which messenger RNA prepared from the postmortem Alzheimers disease (AD) brain can be used for the successful preparation of a recombinant cDNA library. Initial experiments focused on the glial-specific marker glial fibrillary acidic protein (GFAP) since GFAP expression appeared to be a model for further studies on mRNAs that may continue to be expressed at high levels in the vicinity of lesioned sites in the AD brain. An AD cDNA library, prepared in the lambda gt11 expression vector system contained GFAP-specific recombinants. One of these was sequenced and the insert was shown to exhibit 88% homology with the similar sequence from mouse GFAP. As established by Northern blots, the size of the GFAP mRNA prepared from the routinely acquired postmortem AD cortex, approximately 2.7 kb, was the same as from a neurologically normal control brain. These results agree with earlier studies on GFAP mRNA from fresh mouse brain. The results demonstrate that in the postmortem AD brain, astroglial-specific mRNA remains sufficiently stable for molecular genetic analysis and may serve as a useful model for examining the genetic expression of mRNAs that may be related to the molecular pathogenesis and the etiology of AD.

ITC Analysis of Ligand Binding to PreQ1 Riboswitches

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.

Interplay between cystic fibrosis transmembrane regulator and gap junction channels made of connexins 45, 40, 32 and 50 expressed in oocytes.

The cystic fibrosis transmembrane regulator (CFTR) is a Cl− channel known to influence other channels, including connexin (Cx) channels. To study the functional interaction between CFTR and gap junction channels, we coexpressed in Xenopus oocytes CFTR and either Cx45, Cx40, Cx32 or Cx50 and monitored junctional conductance (Gj) and its sensitivity to transjunctional voltage (Vj) by the dual voltage-clamp method. Application of forskolin induced a Cl− current; increased Gj approximately 750%, 560%, 64% and 8% in Cx45, Cx40, Cx32 and Cx50, respectively; and decreased sensitivity to Vj gating, monitored by a change in the ratio between Gj steady state and Gj peak (GjSS/GjPK) at the pulse. In oocyte pairs expressing just Cx45 in one oocyte (#1) and both Cx45 and CFTR in the other (#2), with negative pulses applied to oocyte #1 forskolin application still increased Gj and decreased the sensitivity to Vj gating, indicating that CFTR activation is effective even when it affects only one of the two hemichannels and that the Gj and Vj changes are not artifacts of decreased membrane resistance in the pulsed oocyte. COOH-terminus truncation reduced the forskolin effect on Cx40 (Cx40TR) but not on Cx32 (Cx32TR) channels. The data suggest a cross-talk between CFTR and a variety of gap junction channels. Cytoskeletal scaffolding proteins and/or other intermediate cytoplasmic proteins are likely to play a role in CFTR-Cx interaction.

Observation of preQ1-II riboswitch dynamics using single-molecule FRET

ABSTRACT PreQ1 riboswitches regulate the synthesis of the hypermodified tRNA base queuosine by sensing the pyrrolopyrimidine metabolite preQ1. Here, we use single-molecule FRET to interrogate the structural dynamics of apo and preQ1-bound states of the preQ1-II riboswitch from Lactobacillus rhamnosus. We find that the apo-form of the riboswitch spontaneously samples multiple conformations. Magnesium ions and preQ1 stabilize conformations that sequester the ribosome-binding site of the mRNA within the pseudoknotted structure, thus inhibiting translation initiation. Our results reveal that folding of the preQ1-II riboswitch is complex and provide evidence favoring a conformational selection model of effector binding by riboswitches of this class.

Metal Dependence of Ligand Binding and Heavy-Atom Derivatization of Evolutionarily Distinct PreQ1 Riboswitches

Riboswitches are functional RNA elements located most frequently within the 5′-leader sequences of bacterial mRNAs. By directly binding to small molecules via an aptamer domain, a riboswitch can adapt quickly to changes in the concentration of a specific intracellular ligand, thereby establishing a feedback loop that controls gene expression. Here we discuss methods utilized in the structure determination of evolutionarily distinct classes of preQ1 riboswitches known as class I and II, respectively. These riboswitches “sense” the pyrrolopyrimidine metabolite preQ1—an intermediate on the biosynthetic pathway that produces the hypermodified tRNA base queuosine, which imparts translational fidelity. Herein, we describe (1) the use of isothermal titration calorimetry (ITC) to explore metal ion requirements for ligand binding and (2) modifications to crystallization media containing SO 4 2 − or Na+ that were necessary for phase determination using site-bound Os(NH3) 5 3 + or Cs+ ions, respectively. Our experience has shown that simple manipulations to the mother liquor can lead to favorable binding of the latter ions without the need to engineer metal-binding sites, thus making our methods a first-choice approach that is broadly applicable to functional RNAs.