Arginine-rich motifs present multiple interfaces for specific binding by RNA

Bayer, Travis; Booth, Lauren N.; Knudsen, Scott M.; Ellington, Andrew D.

doi:10.1261/rna.2167605

Cited by 110 publications

(113 citation statements)

References 36 publications

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“…NUMR-1 and NUMR-2 also contain an arginine-rich region near the predicted NLS. This domain is similar to that observed in RNA-binding proteins (Bayer et al, 2005), which suggests that they may function in RNA processing. The presence of a histidine-rich region in NUMR-1 and NUMR-2 suggests that these proteins may chelate transition metals.…”

Section: Discussionsupporting

confidence: 75%

Molecular characterization ofnumr-1andnumr-2: genes that increase both resistance to metal-induced stress and lifespan inCaenorhabditis elegans

Tvermoes

Boyd

Freedman

2010

Journal of Cell Science

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SummaryTo define the mechanisms involved in the molecular response to the carcinogenic metal cadmium, two novel metal-inducible genes from C. elegans were characterized: numr-1 and numr-2 (nuclear localized metal responsive). numr-1 and numr-2 sequences and cellular patterns of expression are identical, indicating that these are functionally equivalent genes. Constitutive transcription of numr-1 and numr-2 is developmentally regulated and occurs in the intestine, in head and tail neurons, and vulva muscles. Exposure to metals induces numr-1 and numr-2 transcription in pharyngeal and intestinal cells. Other environmental stressors do not affect transcription, indicating that these are metal-specific, stress-responsive genes. NUMR-1 and NUMR-2 target to nuclei and colocalize with HSF-1, suggesting that they may be components of nuclear stress granules. Nematodes overexpressing NUMR-1 and NUMR-2 are resistant to stress and live longer than control animals; likewise reducing expression increases sensitivity to metals and decreases neuromuscular functions. Upstream regulatory regions of both genes contain potential binding sites for DAF-16 and SKN-1, which are components of the insulin-IGF-like signaling pathway. This pathway regulates longevity and stress responses in C. elegans. NUMR-1 and NUMR-2 may function to promote resistance to environmental stressors and longevity, which is mediated by the insulin-IGF-like signaling pathway.Key words: C. elegans, Cadmium, Stress response, Lifespan, LongevityJournal of Cell Science increase in the steady-state mRNA level following a 24-hour cadmium exposure (Cui et al., 2007). A second C. elegans gene, originally designated F08F8.1, was subsequently identified based on sequence identity. These genes are now designated numr-1 and numr-2 (nuclear localized metal responsive), respectively.In the present report, the genomic organization, in vivo cellular patterns of expression, effects of transition metals and other stressors on transcription, and phenotypes associated with increased and decreased expression of numr-1 and numr-2 are presented. Phenotypes associated with these genes include changes in neuromuscular activities, as well as increased resistance to metal toxicity and increased lifespan. Results Sequence analysis of numr-1 and numr-2numr-1 and numr-2 were located less than 1 kb apart in a divergent orientation on chromosome III (Fig. 1). numr-1 mRNA was predicted to be encoded by a single exon. By contrast, numr-2 mRNA was predicted to be encoded by five exons, the first of which was ~99% identical to the numr-1 exon. Sequences of the full-length mRNAs and the exon arrangement for numr-1 and numr-2 were determined using 3Ј-and 5Ј-RACE. The sequence of the 3Ј-end of numr-1 was consistent with that reported in WormBase (version WS201). Using 3Ј RACE, however, cDNAs from predicted exons 2-5 of numr-2 were not isolated. This suggested that numr-2 was annotated incorrectly and that it also was a single exon gene. Sequences for the 5Ј-ends of numr-1 and numr-2 were 100% iden...

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Section: Discussionsupporting

confidence: 75%

Molecular characterization ofnumr-1andnumr-2: genes that increase both resistance to metal-induced stress and lifespan inCaenorhabditis elegans

Tvermoes

Boyd

Freedman

2010

Journal of Cell Science

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“…The cross-linking results suggest that these amino acids are concentrated in the N-terminal region but do not exclude the possibility that some RNA contacts are in the middle region. The results presented here indicate that the proposed ARMs are not likely to form direct contacts with the unbranched rodlike RNA and therefore might not function as typical RNAbinding ARMs (62). The observation that HDAg recognizes the RNA secondary structure but not the primary sequence, per se (Griffin and Casey, unpublished), suggests a type of interaction different from those involving most ARMs, which are generally sequence specific (62), and may be more consistent with numerous amino acid contacts in the binding of HDV RNA by HDAg.…”

Section: Discussionmentioning

confidence: 84%

Arginine-Rich Motifs Are Not Required for Hepatitis Delta Virus RNA Binding Activity of the Hepatitis Delta Antigen

Daigh

Griffin

Soroush

et al. 2013

J Virol

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Hepatitis delta virus (HDV) is a unique human pathogen that increases the severity of liver disease in those infected with its helper virus, hepatitis B virus (HBV). The ϳ1,680-nucleotide (nt) single-stranded circular RNA genome assumes a characteristic unbranched rodlike structure in which ϳ70% of the nucleotides form Watson-Crick base pairs (1, 2). Genome replication occurs via a double-rolling-circle mechanism in which host RNA polymerase is redirected to synthesize genomic and antigenomic HDV RNAs, as well as the mRNA for hepatitis delta antigen (HDAg), the sole viral protein (reviewed in references 3 and 4). HDAg forms RNA-protein complexes (RNPs) with both genomic and antigenomic HDV RNAs, and these complexes play essential roles in this unique replication process (5-11). HDAg has been shown to transport HDV RNA to the nucleus, where replication occurs (12). In addition, HDAg interacts with RNA polymerase II (Pol II) (13,14); this interaction may recruit the polymerase to bound HDV RNA and has been proposed to effect changes in the polymerase that permit RNA-directed transcription (14-17). RNPs formed by HDAg and HDV RNA are also packaged into virions (18, 19); these complexes include a modified form of HDAg that interferes with RNA synthesis (20-24). Understanding how HDV RNPs function depends on knowing their structural features, which remains a critical goal.Several studies have attempted to identify regions of HDAg that directly contribute to binding HDV RNA and forming HDV RNPs, but a consistent picture has not yet emerged. In a widely cited model, two arginine-rich motifs (ARM I and ARM II) in the middle region of the protein are thought to form a bipartite RNA binding domain (8). This model is based largely on two in vitro binding studies using bacterially expressed HDAg fusion proteins. One of these studies showed that only fusion proteins containing the middle region of the protein, which includes the ARMs, could bind HDV RNA (25); no RNA binding activity was associated with the N-terminal 78 amino acids (aa). The other study showed loss of in vitro RNA binding activity when either of the ARMs, particularly ARM I, was disrupted by replacement of two basic residues with other amino acids (8). Both reports relied heavily on RNAprotein blots (Northwestern blots), which depend on the ability to remove bound detergent and properly refold proteins following electrophoresis and blotting. In contrast, other in vitro studies implicated the amino-terminal domain of HDAg in binding HDV . However, these analyses were limited by the use of small segments of the protein; Poisson et al. used small peptide fragments (28), and Huang and Wu and Wang et al. employed an N-terminal 88-aa region that bound equally well to HDV and non-HDV RNAs (26,27).Analyses of HDAg-HDV RNA interactions in cells have also yielded conflicting interpretations. Wang et al. (11) found that mutation of ARM I severely diminished the ability of HDAg to package the RNA into secreted particles, a result that could suggest a role for ARM I in ...

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“…In these images, the SRP Alu domain is located in the elongation factor-binding site and the region of SRP9 comprising the key residues is in proximity of h5 and h15 of 18S ribosomal RNA. Lysine usually makes nonspecific electrostatic interactions with the phosphate backbone of nucleic acids, whereas arginine has the additional ability to engage in diverse ionic, weakly polar, and van der Waals interactions as well as in hydrogen bonding with the phosphate backbone and the base (Bayer et al 2005). Since the critical residues in SRP9/14 can all be lysines, we hypothesize that the putative interactions with the ribosomal RNA are mediated by nonbase-specific electrostatic contacts.…”

Section: Discussionmentioning

confidence: 98%

Residues in SRP9/14 essential for elongation arrest activity of the signal recognition particle define a positively charged functional domain on one side of the protein

Mary

Scherrer²,

Huck³

et al. 2010

RNA

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The signal recognition particle (SRP) is a ubiquitous cytoplasmic ribonucleoprotein complex required for the cotranslational targeting of proteins to the endoplasmic reticulum (ER). In eukaryotes, SRP has to arrest the elongation of the nascent chains during targeting to ensure efficient translocation of the preprotein, and this function of SRP is dependent on SRP9/14. Here we present the results of a mutational study on the human protein h9/14 that identified and characterized regions and single residues essential for elongation arrest activity. Effects of the mutations were assessed both in cell-free translation/translocation assays and in cultured mammalian cells. We identified two patches of basic amino acid residues that are essential for activity, whereas the internal loop of SRP14 was found to be dispensable. One patch of important basic residues comprises the previously identified basic pentapetide KRDKK, which can be substituted by four lysines without loss of function. The other patch includes three lysines in the solvent-accessible a2 of h9. All essential residues are located in proximity in SRP9/14 and their basic character suggests that they serve as a positively charged platform for interactions with ribosomal RNA. In addition, they can all be lysines consistent with the hypothesis that they recognize their target(s) via electrostatic contacts, most likely with the phosphate backbone, as opposed to contacts with specific bases.

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Arginine-rich motifs present multiple interfaces for specific binding by RNA

Cited by 110 publications

References 36 publications

Molecular characterization ofnumr-1andnumr-2: genes that increase both resistance to metal-induced stress and lifespan inCaenorhabditis elegans

Molecular characterization ofnumr-1andnumr-2: genes that increase both resistance to metal-induced stress and lifespan inCaenorhabditis elegans

Arginine-Rich Motifs Are Not Required for Hepatitis Delta Virus RNA Binding Activity of the Hepatitis Delta Antigen

Residues in SRP9/14 essential for elongation arrest activity of the signal recognition particle define a positively charged functional domain on one side of the protein

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