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Thermodynamic and phylogenetic insights into hnRNP A1 recognition of the HIV-1 exon splicing silencer 3 element.
Biochemistry. 2014 Apr 08; 53(13):2172-84.B

Abstract

Complete expression of the HIV-1 genome requires balanced usage of suboptimal splice sites. The 3' acceptor site A7 (ssA7) is negatively regulated in part by an interaction between the host hnRNP A1 protein and a viral splicing silencer (ESS3). Binding of hnRNP A1 to ESS3 and other upstream silencers is sufficient to occlude spliceosome assembly. Efforts to understand the splicing repressive properties of hnRNP A1 on ssA7 have revealed hnRNP A1 binds specific sites within the context of a highly folded RNA structure; however, biochemical models assert hnRNP A1 disrupts RNA structure through cooperative spreading. In an effort to improve our understanding of the ssA7 binding properties of hnRNP A1, herein we have performed a combined phylogenetic and biophysical study of the interaction of its UP1 domain with ESS3. Phylogenetic analyses of group M sequences (x̅ = 2860) taken from the Los Alamos HIV database reveal the ESS3 stem loop (SL3(ESS3)) structure has been conserved throughout HIV-1 evolution, despite variations in primary sequence. Calorimetric titrations with UP1 clearly show the SL3(ESS3) structure is a critical binding determinant because deletion of the base-paired region reduces the affinity by ∼150-fold (Kd values of 27.8 nM and 4.2 μM). Cytosine substitutions of conserved apical loop nucleobases show UP1 preferentially binds purines over pyrimidines, where site-specific interactions were detected via saturation transfer difference nuclear magnetic resonance. Chemical shift mapping of the UP1-SL3(ESS3) interface by (1)H-(15)N heteronuclear single-quantum coherence spectroscopy titrations reveals a broad interaction surface on UP1 that encompasses both RRM domains and the inter-RRM linker. Collectively, our results describe a UP1 binding mechanism that is likely different from current models used to explain the alternative splicing properties of hnRNP A1.

Authors+Show Affiliations

Department of Chemistry, Case Western Reserve University , Cleveland, Ohio 44106-7078, United States.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, N.I.H., Extramural

Language

eng

PubMed ID

24628426

Citation

Rollins, Carrie, et al. "Thermodynamic and Phylogenetic Insights Into hnRNP A1 Recognition of the HIV-1 Exon Splicing Silencer 3 Element." Biochemistry, vol. 53, no. 13, 2014, pp. 2172-84.
Rollins C, Levengood JD, Rife BD, et al. Thermodynamic and phylogenetic insights into hnRNP A1 recognition of the HIV-1 exon splicing silencer 3 element. Biochemistry. 2014;53(13):2172-84.
Rollins, C., Levengood, J. D., Rife, B. D., Salemi, M., & Tolbert, B. S. (2014). Thermodynamic and phylogenetic insights into hnRNP A1 recognition of the HIV-1 exon splicing silencer 3 element. Biochemistry, 53(13), 2172-84. https://doi.org/10.1021/bi500180p
Rollins C, et al. Thermodynamic and Phylogenetic Insights Into hnRNP A1 Recognition of the HIV-1 Exon Splicing Silencer 3 Element. Biochemistry. 2014 Apr 8;53(13):2172-84. PubMed PMID: 24628426.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Thermodynamic and phylogenetic insights into hnRNP A1 recognition of the HIV-1 exon splicing silencer 3 element. AU - Rollins,Carrie, AU - Levengood,Jeffrey D, AU - Rife,Brittany D, AU - Salemi,Marco, AU - Tolbert,Blanton S, Y1 - 2014/03/26/ PY - 2014/3/18/entrez PY - 2014/3/19/pubmed PY - 2014/6/24/medline SP - 2172 EP - 84 JF - Biochemistry JO - Biochemistry VL - 53 IS - 13 N2 - Complete expression of the HIV-1 genome requires balanced usage of suboptimal splice sites. The 3' acceptor site A7 (ssA7) is negatively regulated in part by an interaction between the host hnRNP A1 protein and a viral splicing silencer (ESS3). Binding of hnRNP A1 to ESS3 and other upstream silencers is sufficient to occlude spliceosome assembly. Efforts to understand the splicing repressive properties of hnRNP A1 on ssA7 have revealed hnRNP A1 binds specific sites within the context of a highly folded RNA structure; however, biochemical models assert hnRNP A1 disrupts RNA structure through cooperative spreading. In an effort to improve our understanding of the ssA7 binding properties of hnRNP A1, herein we have performed a combined phylogenetic and biophysical study of the interaction of its UP1 domain with ESS3. Phylogenetic analyses of group M sequences (x̅ = 2860) taken from the Los Alamos HIV database reveal the ESS3 stem loop (SL3(ESS3)) structure has been conserved throughout HIV-1 evolution, despite variations in primary sequence. Calorimetric titrations with UP1 clearly show the SL3(ESS3) structure is a critical binding determinant because deletion of the base-paired region reduces the affinity by ∼150-fold (Kd values of 27.8 nM and 4.2 μM). Cytosine substitutions of conserved apical loop nucleobases show UP1 preferentially binds purines over pyrimidines, where site-specific interactions were detected via saturation transfer difference nuclear magnetic resonance. Chemical shift mapping of the UP1-SL3(ESS3) interface by (1)H-(15)N heteronuclear single-quantum coherence spectroscopy titrations reveals a broad interaction surface on UP1 that encompasses both RRM domains and the inter-RRM linker. Collectively, our results describe a UP1 binding mechanism that is likely different from current models used to explain the alternative splicing properties of hnRNP A1. SN - 1520-4995 UR - https://www.unboundmedicine.com/medline/citation/24628426/Thermodynamic_and_phylogenetic_insights_into_hnRNP_A1_recognition_of_the_HIV_1_exon_splicing_silencer_3_element_ L2 - https://doi.org/10.1021/bi500180p DB - PRIME DP - Unbound Medicine ER -