SAFA: Semi-automated footprinting analysis software for high-throughput quantification of nucleic acid footprinting experiments

Das, Rhiju; Laederach, Alain; Pearlman, Samuel M.; Herschlag, Daniel; Altman, Russ B.

doi:10.1261/rna.7214405

Cited by 303 publications

(328 citation statements)

References 42 publications

(58 reference statements)

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“…Reactions (4 min at 25°C) were quenched by adding 11 l of stop solution [0.1 M thiourea͞73% (vol͞vol) formamide͞81 mM Tris-borate͞1.8 mM EDTA͞marker dyes]. Sites of cleavage were resolved by denaturing gel electrophoresis (15% polyacrylamide͞7 M urea) and intensities for individual bands were obtained by integration (32). Background was established in control experiments performed with the 310-BABE and 336-ITE constructs, in which reagents or tethering components were omitted.…”

Section: Methodsmentioning

confidence: 99%

Structure of an RNA switch that enforces stringent retroviral genomic RNA dimerization

Badorrek

Gherghe²,

Weeks³

2006

Proc. Natl. Acad. Sci. U.S.A.

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Retroviruses selectively package two copies of their RNA genomes in the context of a large excess of nongenomic RNA. Specific packaging of genomic RNA is achieved, in part, by recognizing RNAs that form a poorly understood dimeric structure at their 5 ends. We identify, quantify the stability of, and use extensive experimental constraints to calculate a 3D model for a tertiary structure domain that mediates specific interactions between RNA genomes in a gamma retrovirus. In an initial interaction, two stem-loop structures from one RNA form highly stringent crossstrand loop-loop base pairs with the same structures on a second genomic RNA. Upon subsequent folding to the final dimer state, these intergenomic RNA interactions convert to a high affinity and compact tertiary structure, stabilized by interdigitated interactions between U-shaped RNA units. This retroviral conformational switch model illustrates how two-step formation of an RNA tertiary structure yields a stringent molecular recognition event at early assembly steps that can be converted to the stable RNA architecture likely packaged into nascent virions. retroviral RNA dimer ͉ RNA folding ͉ Selective 2Ј-Hydroxyl Acylation analyzed by Primer Extension (SHAPE) chemistry ͉ site-directed cleavage R etroviral genomes usually consist of two sense-strand RNAs that are noncovalently linked near their 5Ј ends to form a dimeric structure (1-3). Recognition of this dimeric state ensures that exactly two RNA genomes are packaged into each nascent virion (1, 2). Mature retroviral virions contain almost exclusively retroviral genomic RNA plus a few select cellular RNAs (4, 5). Many other cellular RNAs, including mRNAs (6-8), are accessible to the retroviral packaging process. Specific recognition of retroviral genomic RNA against a large background of cellular RNA thus represents a striking example of molecular recognition in biology.We have recently identified a minimal dimerization active sequence (MiDAS) (9) for a representative gamma retrovirus, the Moloney murine sarcoma virus (MuSV; Fig. 1A). The MiDAS domain correlates closely with retroviral genomic sequences sufficient to package heterologous RNAs into virions (6,8,11,12), as dimers (8). The MiDAS domain also includes conserved sequence elements previously proposed to specify the noncovalent interactions that mediate RNA dimerization.Conserved sequence elements include self-complementary (palindromic) sequences (PAL1 and PAL2) and stem-loop structures 1 and 2 (SL1 and SL2) (10, 13-16). SL1 and SL2 contain GACG tetraloops that form stable loop-loop interactions with a second RNA molecule. Loop-loop interactions are mediated by canonical intermolecular C-G base pairing and additional stacking and intraand intermolecular hydrogen bonds (17) (see Fig. 6, which is published as supporting information on the PNAS web site). In addition, the self-complementary PAL1 and PAL2 sequences form extended heteroduplexes involving both strands in the dimer (refs. 13-16; C.S.B. and K.M.W., unpublished data). However, the kine...

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Section: Methodsmentioning

confidence: 99%

Structure of an RNA switch that enforces stringent retroviral genomic RNA dimerization

Badorrek

Gherghe²,

Weeks³

2006

Proc. Natl. Acad. Sci. U.S.A.

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“…For quantitative analysis of cleavage bands, SAFA (SemiAutomated Footprinting Analysis) software (Das et al 2005;Laederach et al 2008) was used as described previously (Kafasla et al 2010). To compensate for possible variations in loading, the band intensity values generated by the SAFA software were first normalized with respect to background bands, whose intensity was largely invariant in all lanes (including the Cys-less control lane) but sometimes fluctuated in a few lanes in parallel with each other (and with the full-length primer extension product when it was visible).…”

Section: Tethered Hydroxyl Radical Probing Assaysmentioning

confidence: 99%

Activation of picornaviral IRESs by PTB shows differential dependence on each PTB RNA-binding domain

Kafasla¹,

Lin²,

Curry³

et al. 2011

RNA

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Polypyrimidine tract binding protein (PTB) is an RNA-binding protein with four RNA-binding domains (RBDs). It is a major regulator of alternative splicing and also stimulates translation initiation at picornavirus IRESs (internal ribosome entry sites). The sites of interaction of each RBD with two picornaviral IRESs have previously been mapped. To establish which RBD-IRES interactions are essential for IRES activation, point mutations were introduced into the RNA-binding surface of each RBD. Three such mutations were sufficient to inactivate RNA-binding by any one RBD, but the sites of the other three RBD-IRES interactions remained unperturbed. Poliovirus IRES activation was abrogated by inactivation of RBD1, 2, or 4, but the RBD3-IRES interaction was superfluous. Stimulation of the encephalomyocarditis virus IRES was reduced by inactivation of RBD1, 3, or 4, and abrogated by mutation of RBD2, or both RBDs 3 and 4. Surprisingly, therefore, the binding of PTB in its normal orientation does not guarantee IRES activation; three native RBDs are sufficient for correct binding but not for activation if the missing RBD-IRES interaction is critical.

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“…Thus, a large body of kinetic data on RNA folding at the residue-level is readily obtainable. This large body of data can be analyzed in minutes instead of in hours using freely available semi-automated software (https://simtk.org/ home/safa) [110]. Still higher-throughput data acquisition is now possible using capillary electrophoresis and a software analysis package called CAFA [111].…”

Section: Rna: Present and Futurementioning

confidence: 99%

Unwinding RNA's secrets: advances in the biology, physics, and modeling of complex RNAs

Chu

Herschlag

2008

Current Opinion in Structural Biology

Self Cite

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SummaryThe rapid development of our understanding of the diverse biological roles fulfilled by non-coding RNA has motivated interest in the basic macromolecular behavior, structure, and function of RNA. We focus on two areas in the behavior of complex RNAs. First, we present advances in the understanding of how RNA folding is accomplished in vivo by presenting a mechanism for the action of DEAD-box proteins. Members of this family are intimately associated with almost all cellular processes involving RNA, mediating RNA structural rearrangements and chaperoning their folding. Next, we focus on advances in understanding and characterizing the basic biophysical forces that govern the folding of complex RNAs. Ultimately we expect that a confluence and synergy between these approaches will lead to profound understanding of RNA and its biology.The explosion in our knowledge about noncoding RNA (ncRNA) -RNA that is not translated into protein -has expanded interest in RNA beyond the traditional RNA community. Diverse ncRNAs include the recently-discovered riboswitches, whose novel gene-regulation function is induced by the binding of small metabolites [1]; most of the so-called "Human Accelerated Regions" (HAR) of the human genome, whose recent evolution may be linked to the emergence of the human brain [2]; and many ncRNAs of unknown function [3]. These discoveries underscore the need to understand the fundamental macromolecular behavior of RNA, its structure and dynamics, and how these RNAs are handled and exploited in biology.Study of catalytic RNAs, which allow detection of correct folding via activity measurements, has established basic thermodynamic and kinetic properties of RNA folding. Large catalytic RNAs such as the group I intron from Tetrahymena thermophila and the RNase P RNA from Bacillus subtilis fold at vastly different rates under different conditions upon addition of Mg 2+ and have been shown to fold via multiple parallel pathways, in which different molecules follow different routes with different rates and intermediates, to a common final folded structure [4,5]. These observations support the common view that RNAs traverse rugged energy landscapes as they fold [6,7]. However, little is known about the true shape of these

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SAFA: Semi-automated footprinting analysis software for high-throughput quantification of nucleic acid footprinting experiments

Cited by 303 publications

References 42 publications

Structure of an RNA switch that enforces stringent retroviral genomic RNA dimerization

Structure of an RNA switch that enforces stringent retroviral genomic RNA dimerization

Activation of picornaviral IRESs by PTB shows differential dependence on each PTB RNA-binding domain

Unwinding RNA's secrets: advances in the biology, physics, and modeling of complex RNAs

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