Determining the defining lengths between mature microRNAs/small interfering RNAs and tinyRNAs.

MicroRNAs (miRNAs) and small interfering RNAs (siRNAs) are loaded into Argonaute (AGO) proteins, forming RNA-induced silencing complexes (RISCs). The assembly process establishes the seed, central, 3' supplementary, and tail regions across the loaded guide, enabling the RISC to recognize target RNAs for silencing. This guide segmentation is caused by anchoring the 3' end at the AGO PAZ domain, but the minimum guide length required for the conformation remains to be studied because the current miRNA size defined by Dicer processing is ambiguous. Using a 3' → 5' exonuclease ISG20, we determined the lengths of AGO-associated miR-20a and let-7a with 3' ends that no longer reach the PAZ domain. Unexpectedly, miR-20a and let-7a needed different lengths, 19 and 20 nt, respectively, to maintain their RISC conformation. This difference can be explained by the low affinity of the PAZ domain for the adenosine at g19 of let-7a, suggesting that the tail-region sequence slightly alters the minimum guide length. We also present that 17-nt guides are sufficiently short enough to function as tinyRNAs (tyRNAs) whose 3' ends are not anchored at the PAZ domain. Since tyRNAs do not have the prerequisite anchoring for the standardized guide segmentation, they would recognize targets differently from miRNAs and siRNAs.

Determining the defining lengths between mature microRNAs/small interfering RNAs and tinyRNAs.

MicroRNAs (miRNAs) and small interfering RNAs (siRNAs) are loaded into Argonaute (AGO) proteins, forming RNA-induced silencing complexes (RISCs). The assembly process establishes the seed, central, 3' supplementary, and tail regions across the loaded guide, enabling the RISC to recognize and cleave target RNAs. This guide segmentation is caused by anchoring the 3' end at the AGO PAZ domain, but the minimum guide length required for the conformation remains to be studied because there was no method by which to do so. Using a 3'→5' exonuclease ISG20, we determined the lengths of AGO-associated miR-20a and let-7a with 3' ends that no longer reach the PAZ domain. Unexpectedly, miR-20a and let-7a needed different lengths, 19 and 20 nt, respectively, to maintain their RISC conformation. This difference can be explained by the low affinity of the PAZ domain for the adenosine at g19 of let-7a, suggesting that the tail-region sequence slightly alters the minimum guide length. We also present that 17-nt guides are sufficiently short enough to function as tinyRNAs (tyRNAs) whose 3' ends are not anchored at the PAZ domain. Since tyRNAs do not have the prerequisite anchoring for the standardized guide segmentation, they would recognize targets differently from miRNAs and siRNAs.

Manganese-dependent microRNA trimming by 3'→5' exonucleases generates 14-nucleotide or shorter tiny RNAs.

MicroRNAs (miRNAs) are about 22-nucleotide (nt) noncoding RNAs forming the effector complexes with Argonaute (AGO) proteins to repress gene expression. Although tiny RNAs (tyRNAs) shorter than 19 nt have been found to bind to plant and vertebrate AGOs, their biogenesis remains a long-standing question. Here, our in vivo and in vitro studies show several 3'→5' exonucleases, such as interferon-stimulated gene 20 kDa (ISG20), three prime repair exonuclease 1 (TREX1), and ERI1 (enhanced RNAi, also known as 3'hExo), capable of trimming AGO-associated full-length miRNAs to 14-nt or shorter tyRNAs. Their guide trimming occurs in a manganese-dependent manner but independently of the guide sequence and the loaded four human AGO paralogs. We also show that ISG20-mediated guide trimming makes Argonaute3 (AGO3) a slicer. Given the high Mn2+ concentrations in stressed cells, virus-infected cells, and neurodegeneration, our study sheds light on the roles of the Mn2+-dependent exonucleases in remodeling gene silencing.

In vitro antibacterial activity of cefiderocol against recent multidrug-resistant carbapenem-nonsusceptible Enterobacterales isolates.

Cefiderocol, a siderophore-containing cephalosporin with broad-spectrum antimicrobial activity against many ß-lactam-resistant Gram-negative bacteria, was tested by broth microdilution against 104 carbapenem-non-susceptible Enterobacterales clinical isolates from 2011 to 2018. Carbapenemase identification was determined using PCR followed by targeted gene sequencing or whole genome sequencing (WGS). All isolates were multidrug-resistant, 89.4% (93/104) and produced a serine (KPC or SME) carbapenemase, with as many as four ß-lactamases present. A VIM-1 or NDM-1 metallo-ß-lactamase was confirmed in 6.7% of the isolates (N = 5 and 2, respectively). All isolates were susceptible to cefiderocol, unlike the comparator agents. Susceptibility for comparators ranged from 24.0% for meropenem to 91.3%, 92.3% and 96.1% for imipenem-relebactam, ceftazidime-avibactam and meropenem-vaborbactam, respectively; 48.1%, 75.2% and 79.8% of the isolates were susceptible to omadacycline, colistin and eravacycline, respectively. Two isolates with cefiderocol MICs of 2 mg/L had mutations or deletions of the iron transport genes fhuA/E or fepA, as determined by WGS.