An Evolved RNA Recognition Motif That Suppresses HIV-1 Tat/TAR-Dependent Transcription

Abstract

Potent and selective recognition and modulation of disease-relevant RNAs remains a daunting challenge. We previously examined the utility of the U1A N-terminal RNA recognition motif as a scaffold for tailoring new RNA hairpin recognition, and showed that as few as one or two mutations can result in moderate affinity (low μM dissociation constant) for the human immunodeficiency virus (HIV) Trans-Activation Response element (TAR) RNA, an RNA hairpin controlling transcription of the human immunodeficiency virus (HIV) genome. Here we use yeast display and saturation mutagenesis of established RNA binding regions in U1A to identify new synthetic proteins that potently and selectively bind TAR RNA. Our best candidate has truly altered, not simply broadened, RNA binding selectivity; it binds TAR with sub-nanomolar affinity (apparent dissociation constant ~0.5 nM), but does not appreciably bind the original U1A RNA target (U1hpII). It specifically recognizes the TAR RNA hairpin in the context of the HIV-1 5′-untranslated region, inhibits the interaction between TAR RNA and an HIV Trans-activator of transcription (Tat)-derived peptide, and suppresses Tat/TAR-dependent transcription. Proteins described in this work are among the tightest TAR RNA-binding reagents – small molecule, nucleic acid, or protein - reported to date, and thus have potential utility as therapeutics and basic research tools. Moreover, our findings demonstrate how a naturally occurring RNA recognition motif can be dramatically resurfaced through mutation, leading to potent and selective recognition—and modulation—of a disease-relevant RNA.

Face-time with TAR: Portraits of an HIV-1 RNA with diverse modes of effector recognition relevant for drug discovery

“…During the past 2 decades, multiple labs have worked to identify small molecules that bind HIV-1 TAR (64,(85)(86)(87)(88)(89)(90)(91). To gain perspective about the successes and ongoing challenges, it is instructive to examine the handful of structurally characterized TAR-small molecule complexes to assess compound localization and commonalities in their modes of molecular recognition.…”

“…Advances in protein engineering have facilitated the design of novel RNA-binding proteins with distinct functions (134). In this regard, the TAR-binding protein (TBP) is a model system that was "evolved" from RRM1 of the U1A spliceosomal protein by combining saturation mutagenesis, yeast display, and cell sorting (77,85). The unique mode of HIV-1 TAR recognition by variant TBP6.7 was visualized recently by a co-crystal structure determined to 1.80 Å resolution (77).…”

Amiloride as a new RNA-binding scaffold with activity against HIV-1 TAR

“…17 Importantly, this thienopyridine-based probe was charge neutral and bound TAR with a dissociation constant of 2.4 μM. Finally, branched and cyclic peptides 58-60 as well as small synthetic proteins 61 have also shown promise as potent inhibitors of the TAR:Tat interaction. In order to expedite screening and increase hit rates, the Al-Hashimi lab recently pioneered a virtual docking method for RNA by first generating dynamic ensembles of TAR RNA through molecular dynamics (MD) simulations and NMR spectroscopy refinements.…”

The potential of engineered eukaryotic RNA‐binding proteins as molecular tools and therapeutics

“…This challenge can potentially be overcome with high throughput screens or in vitro evolution methods to select the desired specificity. For the latter, phage display has been used on the U1A RRM to tailor RNA‐binding to the TAR RNA (Crawford et al, ). The second method for RBD design is to use PUF and PPR domains and their established recognition codes to engineer novel RBPs to bind specific sequences of interest.…”