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Jacks, Amanda; Kelly, Geoff; Curry, Stephen; Conte, Maria R.

doi:10.1023/a:1014928117895

Cited by 5 publications

(2 citation statements)

References 6 publications

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“…A CryoProbe™ is recommended to increase instrument sensitivity. As an additional quality control, the La antigen conformation can be verified by comparing to reference spectra (BMRB ID: 5235) (Jacks et al, 2002). …”

Section: Basic Protocol 2 - Alarm Nmr Counter-screenmentioning

confidence: 99%

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ALARM NMR for HTS Triage and Chemical Probe Validation

Dahlin

Cuellar

Singh

et al. 2018

CP Chemical Biology

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Nonspecific target engagement by test compounds and purported chemical probes is a significant source of assay interference and promiscuous bioactivity in high-throughput screening (HTS) and chemical biology. Most counter-screens for thiol-reactive compounds utilize mass spectrometry or fluorescence detection, and non-proteinaceous reporters like glutathione that may not always approximate the reactivity of protein side-chains. By contrast, a La assay to detect reactive molecules by nuclear magnetic resonance (ALARM NMR) is an industry-developed protein-based [H-C]-heteronuclear multiple quantum coherence (HMQC) NMR counter-screen to identify nonspecific protein interactions by test compounds by reporting their tendencies to modulate the human La antigen conformation. This is a users-guide to the production of theC-labeled La antigen reporter protein, the reaction of test compounds with this reporter protein, as well as the collection and analysis of characteristic NMR spectra. Combined with other assay interference counter-screens, this assay will enhance chemical biology by helping researchers better prioritize chemical matter and which will increase the number of tractable HTS screening actives and aid in the development of better chemical probes.

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Section: Basic Protocol 2 - Alarm Nmr Counter-screenmentioning

confidence: 99%

“…As an additional quality control, the La antigen conformation can be verified by comparing to reference spectra (BMRB ID: 5235;Jacks, Kelly, Curry, & Conte, 2002).…”

Section: Incubation Of Test Compounds and Alarm Nmr Proteinmentioning

confidence: 99%

ALARM NMR for HTS Triage and Chemical Probe Validation

Dahlin

Cuellar

Singh

et al. 2018

CP Chemical Biology

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The La and related RNA‐binding proteins (LARPs): structures, functions, and evolving perspectives

et al. 2017

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La was first identified as a polypeptide component of ribonucleic protein (RNP) complexes targeted by antibodies in autoimmune patients and is now known to be a eukaryote cell-ubiquitous protein. Structure and function studies have shown that La binds to a common terminal motif, UUU-3’OH, of nascent RNA polymerase III (RNAP III) transcripts and protects them from exonucleolytic decay. For precursor-tRNAs, the most diverse and abundant of these transcripts, La also functions as a RNA chaperone that helps to prevent their misfolding. Related to this, we review evidence that suggests that La and its link to RNAP III were significant in the great expansions of the tRNAomes that occurred in eukaryotes. Four families of La-related proteins (LARPs) emerged during eukaryotic evolution with specialized functions. We provide an overview of the high resolution structural biology of La and LARPs. LARP7 family members most closely resemble La but function with a single RNAP III nuclear transcript, 7SK or telomerase RNA. A cytoplasmic isoform of La protein as well as LARPs 6, 4 and 1 function in mRNA metabolism and translation in distinct but similar ways, sometimes with the poly(A)-binding protein (PABP), and in some cases by direct binding to poly(A)-RNA. New structures of LARP domains, some complexed with RNA, provide novel insights into the functional versatility of these proteins. We also consider LARPs in relation to ancestral La protein and potential retention of links to specific RNA-related pathways. One such link may be tRNA surveillance and codon usage by LARP associated mRNAs.

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Backbone and side chain NMR assignment, along with the secondary structure prediction of RRM2 domain of La protein from a lower eukaryote exhibiting identical structural organization with its human homolog

et al. 2014

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The La protein (Lupus antigen), a key mediator during biogenesis of RNA polymerase III transcripts, contains a characteristic La motif and one or two RNA recognition motif (RRM) domains, depending on the organism of origin. The RRM1 domain is conserved in higher eukaryotes and located in the N-terminal region, whereas the C-terminal RRM2 domain is absent in most lower eukaryotes and its specific role remains, so far, uncharacterized. Here, we present the backbone and side-chain assignment of the (1)H, (13)C and (15)N resonances of RRM2 of La protein from Dictyostelium discoideum. Interestingly, the La protein in this lower eukaryote, exhibits high homology to its human counterpart. Moreover, it contains two RRM domains, instead of one, raising questions on its evolutionary origin and the putative role of RRM2 in vivo. We also provide its secondary structure as predicted by the TALOS+ online tool.

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Untitled

Cited by 5 publications

References 6 publications

ALARM NMR for HTS Triage and Chemical Probe Validation

ALARM NMR for HTS Triage and Chemical Probe Validation

The La and related RNA‐binding proteins (LARPs): structures, functions, and evolving perspectives

Backbone and side chain NMR assignment, along with the secondary structure prediction of RRM2 domain of La protein from a lower eukaryote exhibiting identical structural organization with its human homolog

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