Sequence-Specific RNA Binding by a Nova KH Domain

106

The noncoding RNA Xist has been shown to be essential for X-chromosome inactivation and to coat the inactive X-chromosome (Xi). Thus, an important question in understanding the formation of Xi is whether the binding reaction of Xist is necessary for X-chromosome inactivation. In this article, we demonstrate the failure of X-chromosome silencing if the association of Xist with the X-chromosome is inhibited. The chromatin-binding region was functionally mapped and evaluated by using an approach for studying noncoding RNA function in living cells that we call peptide nucleic acid (PNA) interference mapping. In the reported experiments, a single 19-bp antisense cell-permeating PNA targeted against a particular region of Xist RNA caused the disruption of the Xi. The association of the Xi with macro-histone H2A is also disturbed by PNA interference mapping.

Section: Resultsmentioning

confidence: 71%

“…Several lines of evidence, including x-ray crystallographic analysis, show that the NOVA2͞NOVA1-binding site is UCAY, where Y stands for a pyrimidine. Furthermore, the sequence GAGUCAU was shown to be an optimal binding site for the NOVA2 protein by in vitro selection (SELEX) experiments (34)(35)(36).…”

Section: Resultsmentioning

confidence: 99%

PNA interference mapping demonstrates functional domains in the noncoding RNA Xist

Beletskii

Hong²,

Pehrson³

et al. 2001

106

“…In vitro RNA selection experiments identified RNAs harboring UCAU elements to which Nova proteins bound with sequence specificity and high affinity (26,27). In addition, the cocrystal structure of Nova protein bound to a UCAY RNA element has been solved and confirms the specific nature of the Nova-RNA interaction (28,29). The Nova-1 RNA selection consensus sequence matched an intronic UCAU repeat element in glycine receptor ␣2 (GlyR␣2) pre-mRNA and identified it as a candidate Nova target.…”

mentioning

confidence: 72%

“…The cocrystal structure of a Nova-RNA complex reveals that most of the hnRNPK homology (KH) domain is accessible for protein-protein interactions, even when bound to RNA (29). Second, Nova contains several proline-and alanine-rich stretches in the spacer region linking the second and third KH domains that may represent protein interaction motifs (27,30).…”

mentioning

confidence: 99%

A brain-enriched polypyrimidine tract-binding protein antagonizes the ability of Nova to regulate neuron-specific alternative splicing

Polydorides

Okano

Yang

et al. 2000

219

189

The Nova paraneoplastic antigens are neuron-specific RNA binding proteins that participate in the control of alternative splicing. We have used the yeast two-hybrid system to isolate Nova interacting proteins and identify an RNA binding protein that is closely related to the polypyrimidine tract-binding protein (PTB). The expression of this protein, brPTB, is enriched in the brain, where it is expressed in glia and neurons. brPTB interacts with Nova proteins in cell lines and colocalizes with Nova within neuronal nuclei. We previously found that Nova binds to a pyrimidine-rich RNA element present upstream of an alternatively spliced exon, E3A, in glycine receptor ␣2 (GlyR␣2) pre-mRNA, and this binding is implicated in Novadependent regulation of splicing. Cotransfection assays with a GlyR␣2 minigene demonstrate that brPTB antagonizes the action of Nova to increase utilization of GlyR␣2 E3A. brPTB binds to a 90-nt GlyR␣2 RNA adjacent to the Nova binding site, but with an affinity that is more than 10-fold lower than Nova. When a putative binding site for brPTB on the GlyR␣2 RNA is mutated, binding is abolished and the inhibitory effect on Nova-dependent exon selection disappears. These results suggest that brPTB is a tissuerestricted RNA binding protein that interacts with and inhibits the ability of Nova to activate exon selection in neurons.N eurons make extensive use of alternative splicing to regulate functional differences in proteins. A wide variety of neurotransmitter receptor activities are regulated by alternative splicing, including NR1 N-methyl-D-aspartate (NMDA) receptor subcellular localization (1) and interaction with neurofilaments (2), the physiology of the glutamate (3) and NMDA (4) receptors, and the ability of agrin to induce clustering of acetylcholine receptors (5). Moreover, several neurologic diseases such as spinal muscular atrophy, amyotrophic lateral sclerosis, and paraneoplastic opsoclonus-myoclonus ataxia (POMA) have been associated with defects in proteins involved in generating the splicing machinery or in the accurate splicing of target .Since the discovery of tissue-specific splicing of the calcitonin͞ calcitonin gene-related peptide (CGRP) transcript in neurons, there has been an extensive search for cis-acting RNA elements and trans-acting RNA binding proteins that mediate neuronspecific splicing. The first example of cis-acting regulatory elements in neuronal pre-mRNAs identified was in calcitonin͞ CGRP pre-mRNA (9), and a number of specific sequences have been identified that are responsible for calcitonin͞CGRP tissuespecific processing (10, 11). Subsequent work identified regulatory sequences near other neuron-specific exons such as the N1 exon of src (12) and a 24-nt exon of the ␥-aminobutyric acid type A receptor ␥2 subunit (13).The identification of trans-acting factors that regulate neuronal splicing has been a greater challenge. Two general mechanisms might account for the way such factors could mediate regulation of neuronal splicing. Brain-specific variants in splicing ...

“…KH domains, like ribosomal protein S1 domains, are found in a variety of nucleic acid-binding proteins, often in multiple copies within one protein (16). Structural evidence suggests that KH domains interact sequence-specifically with four to five consecutive nucleotides in single-stranded regions (17)(18)(19)(20). Comparable structures of S1 domains in complex with nucleic acids are not available.…”

mentioning

confidence: 99%

A high-affinity interaction between NusA and the rrn nut site in Mycobacterium tuberculosis

Arnvig¹,

Pennell²,

Gopal³

et al. 2004

The bacterial NusA protein enhances transcriptional pausing and termination and is known to play an essential role in antitermination. Antitermination is signaled by a nut-like cis-acting RNA sequence comprising boxB, boxA, and boxC. In the present study, we demonstrate a direct, specific high-affinity interaction between the rrn leader nut-like sites and the NusA proteins of Mycobacterium tuberculosis and Escherichia coli. This NusA-RNA interaction relies on the conserved region downstream of boxA, the boxC region, thus demonstrating a key function of this element. We have established an in vivo assay for antitermination in mycobacteria and use this to show that the M. tuberculosis rrn nut-like site enhances transcriptional read-through of untranslated RNA consistent with an antitermination signal within this site. Finally, we present evidence that this NusA-RNA interaction affects transcriptional events further downstream.N usA (N-using substance A) is a highly conserved transcription factor involved in transcriptional pausing, termination, and antitermination (reviewed in ref. 1). The protein is also thought to be essential for the increased elongation rate associated with rrn transcription and to be a constituent of the rrn antitermination complex (2, 3).The rrn antitermination complex ensures that transcription of rRNA is not terminated by Rho despite the nascent transcripts being untranslated and thus naked (4). The complex is believed to include RNA polymerase (RNAP), Nus factors A, B, E, and G, ribosomal protein S4, and possibly one or more additional cellular components (5-10). The antitermination complex is assembled after transcription of a nut-like site (hereafter referred to as nut site for brevity) consisting of boxB, boxA, and boxC within the rrn leader region (1, 4). However, it has been shown that boxA is sufficient to obtain antitermination (7, 11). It is not entirely clear how the assembly proceeds, but evidence suggests that binding of a NusB͞NusE heterodimer to boxA RNA may play a part, whereas the exact roles of NusA, boxB, and boxC remain unclear (6,7,11,12).Bacterial NusA proteins harbor three RNA-binding domains: S1, K homology 1 (KH1), and KH2 as well as an N-terminal domain that interacts with the RNAP (13-15). KH domains, like ribosomal protein S1 domains, are found in a variety of nucleic acid-binding proteins, often in multiple copies within one protein (16). Structural evidence suggests that KH domains interact sequence-specifically with four to five consecutive nucleotides in single-stranded regions (17)(18)(19)(20). Comparable structures of S1 domains in complex with nucleic acids are not available. Based on the NusA structure of Thermotoga maritima, Worbs et al. (15) proposed that the three RNA-binding domains in the NusA protein act together to form an ''extended RNA binding surface'' consistent with the ability to bind RNA with high affinity and specificity. NusA interacts with boxAB (21) as well as with mRNA (22). Although bacterial NusA proteins display a high degree of co...