Degradome sequencing from S. castellii

ABSTRACT: The RNA-induced silencing complex, comprising Argonaute and guide RNA, mediates RNA interference. Here we report the 3.2 M-CM-^E crystal structure of Kluyveromyces Argonaute (KpAGO) fortuitously complexed with guide RNA originating from small-RNA duplexes autonomously loaded by recombinant KpAGO. Despite their diverse sequences, guide-RNA nucleotides 1-8 are positioned similarly, with sequence-independent contacts to bases, phosphates and 2'-hydroxyl groups pre-organizing the backbone of nucleotides 2-8 in a near-A-form conformation. Compared with prokaryotic Argonautes, KpAGO has numerous surface-exposed insertion segments, with a cluster of conserved insertions repositioning the N domain to enable full propagation of guide-target pairing. Compared with Argonautes in inactive conformations, KpAGO has a hydrogen-bond network that stabilizes an expanded and repositioned loop, which inserts an invariant glutamate into the catalytic pocket. Mutation results and analogies to Ribonuclease H indicate that insertion of this glutamate finger completes a universally conserved catalytic tetrad, thereby activating Argonaute for cleavage. High-throughput sequencing of cellular RNAs containing both a 5'-monophosphate group and a poly(A) tail. To identify Argonaute-dependent cleavage tags, sequencing libraries were prepared from M-NM-^Txrn1 and M-NM-^Txrn1 M-NM-^Tago1 strains.

ORGANISM(S): Naumovozyma castellii

SUBMITTER: David Weinberg 

PROVIDER: E-GEOD-37723 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress