Biology subjects

Gebert, L. F. R.

Publications and source records attributed to Gebert, L. F. R..

4 recordsLinked to original sources

bioRxiv2026

Organyl 5'-Phosphates in siRNA Guide Strands: Structure-Function Relationships Governing Anchoring in Argonaute 2 and Metabolic Stability

Efficient siRNA loading into Argonaute2 (AGO2) requires a 5'-phosphate (5'-P) on the guide strand, yet this group is vulnerable to metabolic degradation in vivo. Although numerous chemical mimics of 5'-P have been reported, structural principles governing AGO2 interactions with organyl substituents on the 5'-P remain unclear. Moreover, structural determinants of 5'-P mimic recognition by known degradative enzymes (principally phosphatases and 5'-exonucleases) are also poorly understood. The 5'-P binding site of the AGO2 MID domain contains a stack of aromatic residues (Y527/F811/Y815), presenting a structural basis for augmenting canonical anchoring interactions. Herein, we systematically synthesized and characterized a diverse panel of organyl 5'-phosphates (5'-POR; R = 35 variable substituents) as guide strand 5'-P mimics designed to engage this unique hydrophobic pocket. Among the compounds evaluated, 5'-POR guide strands bearing methyl (Me) or phenylpropargyl (PhPrp) substituents are well-tolerated by AGO2 in cells. Previously uncharacterized 5'-P mimics, including 5'-phosphorothioate (5'-PS), phenylpropargyl 5'-phosphorothioate (5'-PS-PhPrp), and 5'-mesylphosphoramidate (5'-MsPA), maintain comparable AGO2 compatibility. All examined 5'-P mimics are markedly resistant to phosphatase, while 5'-POR variants and 5'-PS-PhPrp are also resistant to 5'-exonuclease degradation due to masking a negative charge of 5'-P. A crystal structure of a 5'-PO-PhPrp guide strand loaded into AGO2 reveals an unexpected network of {pi}-{pi} interactions between the rigid phenylpropargyl group and the targeted hydrophobic pocket of the MID domain. Collectively, these findings expand the functional chemical space of 5'-P mimics and define new modes for metabolically stabilizing the guide strand 5'-end while augmenting AGO2 MID anchoring. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=85 SRC="FIGDIR/small/705631v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@e6f88forg.highwire.dtl.DTLVardef@1c87dfaorg.highwire.dtl.DTLVardef@1c6e68corg.highwire.dtl.DTLVardef@14a1f60_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2024

Structural basis for gene silencing by siRNAs in humans

Small interfering RNAs (siRNAs) guide mRNA cleavage by human Argonaute2 (hAgo2), leading to targeted gene silencing. Despite their laboratory and clinical impact, structural insights into human siRNA catalytic activity remain elusive. Here, we show that disrupting siRNA 3-end binding by hAgo2 accelerates target cleavage and stabilizes its catalytic conformation, enabling detailed structural analysis. A 3.16 [A] global resolution cryo-EM reconstruction reveals that distortion of the siRNA-target duplex at position 6 allows target RNA entry into the catalytic cleft and shifts Lysine-709, a previously unrecognized catalytic residue, into the active site. A pyrimidine at target nucleotide t10 positions another unrecognized catalytic residue, Arginine-710, for optimal cleavage. Expansion of the guide-target duplex major groove docks the scissile phosphate for hydrolysis and subsequent groove compression after position 16 permits target RNAs to exit the catalytic cleft. These findings reveal how hAgo2 catalyzes siRNA target hydrolysis, providing a high-resolution model for therapeutic design.

molecular biology↗

bioRxiv2024

Assembly of the Bacterial Ribosome with Circularly Permuted rRNA

Co-transcriptional assembly is an integral feature of the formation of RNA-protein complexes that mediate translation. For ribosome synthesis, prior studies have indicated that the strict order of transcription of rRNA domains may not be obligatory during bacterial ribosome biogenesis, since a series of circularly permuted rRNAs are viable. In this work, we report the insights into assembly of the bacterial ribosome large subunit (LSU) based on cryo-EM density maps of intermediates that accumulate during in vitro ribosome synthesis using a set of circularly permuted (CiPer) rRNAs. The observed ensemble of twenty-three resolved ribosome large subunit intermediates reveals conserved assembly routes with an underlying hierarchy among cooperative assembly blocks. There are intricate interdependencies for the formation of key structural rRNA helices revealed from the circular permutation of rRNA. While the order of domain synthesis is not obligatory, the order of domain association does appear to proceed with a particular order, likely due to the strong evolutionary pressure on efficient ribosome synthesis. This work reinforces the robustness of the known assembly hierarchy of the bacterial large ribosomal subunit, and offers a coherent view of how efficient assembly of CiPer rRNAs can be understood in that context.

biophysics↗

bioRxiv2018

Beyond the seed: structural basis for supplementary mircoRNA targeting

microRNAs (miRNA) guide Argonaute proteins to mRNAs targeted for repression. Target recognition occurs primarily through the miRNA seed region, composed of guide (g) nucleotides g2-g8. However, nucleotides beyond the seed are also important for some known miRNA-target interactions. Here, we report the structure of human Argonaute2 (Ago2) engaged with a target RNA recognized through both miRNA seed and supplementary (g13-g16) regions. Ago2 creates a \"supplementary chamber\" that accommodates up to 5 miRNA-target base pairs. Seed and supplementary chambers are adjacent to each other, and can be bridged by an unstructured target loop of 1-15 nucleotides. Opening of the supplementary chamber may be constrained by tension in the miRNA 3' tail as increases in miRNA length stabilize supplementary interactions. Contrary to previous reports, we demonstrate optimal supplementary interactions can increase target affinity >20-fold. These results provide a mechanism for extended miRNA-targeting, suggest a function for 3' isomiRs in tuning miRNA targeting specificity, and indicate that supplementary interactions may contribute more to target recognition than is widely appreciated.

biochemistry↗