13 research outputs found
Potent and Selective Inhibition of A‑to‑I RNA Editing with 2′‑<i>O</i>‑Methyl/Locked Nucleic Acid-Containing Antisense Oligoribonucleotides
ADARs
(adenosine deaminases acting on RNA) are RNA editing enzymes
that bind double helical RNAs and deaminate select adenosines (A).
The product inosine (I) is read during translation as guanosine (G),
so such changes can alter codon meaning. ADAR-catalyzed A to I changes
occur in coding sequences for several proteins of importance to the
nervous system. However, these sites constitute only a very small
fraction of known A to I sites in the human transcriptome, and the
significance of editing at the vast majority sites is unknown at this
time. Site-selective inhibitors of RNA editing are needed to advance
our understanding of the function of editing at specific sites. Here
we show that 2′-<i>O</i>-methyl/locked nucleic acid
(LNA) mixmer antisense oligonucleotides are potent and selective inhibitors
of RNA editing on two different target RNAs. These reagents are capable
of binding with high affinity to RNA editing substrates and remodeling
the secondary structure by a strand-invasion mechanism. The potency
observed here for 2′-<i>O</i>-methyl/LNA mixmers
suggests this backbone structure is superior to the morpholino backbone
structure for inhibition of RNA editing. Finally, we demonstrate antisense
inhibition of editing of the mRNA for the DNA repair glycosylase NEIL1
in cultured human cells, providing a new approach to exploring the
link between RNA editing and the cellular response to oxidative DNA
damage
Structural Analysis of Human Argonaute‑2 Bound to a Modified siRNA Guide
Incorporation of
chemical modifications into small interfering
RNAs (siRNAs) increases their metabolic stability and improves their
tissue distribution. However, how these modifications impact interactions
with Argonaute-2 (Ago2), the molecular target of siRNAs, is not known.
Herein we present the crystal structure of human Ago2 bound to a metabolically
stable siRNA containing extensive backbone modifications. Comparison
to the structure of an equivalent unmodified-siRNA complex indicates
that the structure of Ago2 is relatively unaffected by chemical modifications
in the bound siRNA. In contrast, the modified siRNA appears to be
much more plastic and shifts, relative to the unmodified siRNA, to
optimize contacts with Ago2. Structure–activity analysis reveals
that even major conformational perturbations in the 3′ half
of the siRNA seed region have a relatively modest effect on knockdown
potency. These findings provide an explanation for a variety of modification
patterns tolerated in siRNAs and a structural basis for advancing
therapeutic siRNA design
Structure-Guided Control of siRNA Off-Target Effects
Short interfering
RNAs (siRNAs) are promising therapeutics that
make use of the RNA interference (RNAi) pathway, but liabilities arising
from the native RNA structure necessitate chemical modification for
drug development. Advances in the structural characterization of components
of the human RNAi pathway have enabled structure-guided optimization
of siRNA properties. Here we report the 2.3 Ã… resolution crystal
structure of human Argonaute 2 (hAgo2), a key nuclease in the RNAi
pathway, bound to an siRNA guide strand bearing an unnatural triazolyl
nucleotide at position 1 (g1). Unlike natural nucleotides, this analogue
inserts deeply into hAgo2’s central RNA binding cleft and thus
is able to modulate pairing between guide and target RNAs. The affinity
of the hAgo2–siRNA complex for a seed-only matched target was
significantly reduced by the triazolyl modification, while the affinity
for a fully matched target was unchanged. In addition, siRNA potency
for off-target repression was reduced (4-fold increase in IC<sub>50</sub>) by the modification, while on-target knockdown was improved (2-fold
reduction in IC<sub>50</sub>). Controlling siRNA on-target versus
microRNA (miRNA)-like off-target potency by projection of substituent
groups into the hAgo2 central cleft from g1 is a new approach to enhance
siRNA selectivity with a strong structural rationale
Structural and mechanistic basis of the EMC-dependent biogenesis of distinct transmembrane clients.
Membrane protein biogenesis in the endoplasmic reticulum (ER) is complex and failure-prone. The ER membrane protein complex (EMC), comprising eight conserved subunits, has emerged as a central player in this process. Yet, we have limited understanding of how EMC enables insertion and integrity of diverse clients, from tail-anchored to polytopic transmembrane proteins. Here, yeast and human EMC cryo-EM structures reveal conserved intricate assemblies and human-specific features associated with pathologies. Structure-based functional studies distinguish between two separable EMC activities, as an insertase regulating tail-anchored protein levels and a broader role in polytopic membrane protein biogenesis. These depend on mechanistically coupled yet spatially distinct regions including two lipid-accessible membrane cavities which confer client-specific regulation, and a non-insertase EMC function mediated by the EMC lumenal domain. Our studies illuminate the structural and mechanistic basis of EMC's multifunctionality and point to its role in differentially regulating the biogenesis of distinct client protein classes
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The ER membrane protein complex interacts cotranslationally to enable biogenesis of multipass membrane proteins.
The endoplasmic reticulum (ER) supports biosynthesis of proteins with diverse transmembrane domain (TMD) lengths and hydrophobicity. Features in transmembrane domains such as charged residues in ion channels are often functionally important, but could pose a challenge during cotranslational membrane insertion and folding. Our systematic proteomic approaches in both yeast and human cells revealed that the ER membrane protein complex (EMC) binds to and promotes the biogenesis of a range of multipass transmembrane proteins, with a particular enrichment for transporters. Proximity-specific ribosome profiling demonstrates that the EMC engages clients cotranslationally and immediately following clusters of TMDs enriched for charged residues. The EMC can remain associated after completion of translation, which both protects clients from premature degradation and allows recruitment of substrate-specific and general chaperones. Thus, the EMC broadly enables the biogenesis of multipass transmembrane proteins containing destabilizing features, thereby mitigating the trade-off between function and stability
Recommended from our members
The ER membrane protein complex interacts cotranslationally to enable biogenesis of multipass membrane proteins
The endoplasmic reticulum (ER) supports biosynthesis of proteins with diverse transmembrane domain (TMD) lengths and hydrophobicity. Features in transmembrane domains such as charged residues in ion channels are often functionally important, but could pose a challenge during cotranslational membrane insertion and folding. Our systematic proteomic approaches in both yeast and human cells revealed that the ER membrane protein complex (EMC) binds to and promotes the biogenesis of a range of multipass transmembrane proteins, with a particular enrichment for transporters. Proximity-specific ribosome profiling demonstrates that the EMC engages clients cotranslationally and immediately following clusters of TMDs enriched for charged residues. The EMC can remain associated after completion of translation, which both protects clients from premature degradation and allows recruitment of substrate-specific and general chaperones. Thus, the EMC broadly enables the biogenesis of multipass transmembrane proteins containing destabilizing features, thereby mitigating the trade-off between function and stability
Recommended from our members
The ER membrane protein complex interacts cotranslationally to enable biogenesis of multipass membrane proteins.
The endoplasmic reticulum (ER) supports biosynthesis of proteins with diverse transmembrane domain (TMD) lengths and hydrophobicity. Features in transmembrane domains such as charged residues in ion channels are often functionally important, but could pose a challenge during cotranslational membrane insertion and folding. Our systematic proteomic approaches in both yeast and human cells revealed that the ER membrane protein complex (EMC) binds to and promotes the biogenesis of a range of multipass transmembrane proteins, with a particular enrichment for transporters. Proximity-specific ribosome profiling demonstrates that the EMC engages clients cotranslationally and immediately following clusters of TMDs enriched for charged residues. The EMC can remain associated after completion of translation, which both protects clients from premature degradation and allows recruitment of substrate-specific and general chaperones. Thus, the EMC broadly enables the biogenesis of multipass transmembrane proteins containing destabilizing features, thereby mitigating the trade-off between function and stability