Disruption of the β1L Isoform of GABP Reverses Glioblastoma Replicative Immortality in a TERT Promoter Mutation-Dependent Manner

TERT promoter mutations reactivate telomerase, allowing for indefinite telomere maintenance and enabling cellular immortalization. These mutations specifically recruit the multimeric ETS factor GABP, which can form two functionally independent transcription factor species: a dimer or a tetramer. We show that genetic disruption of GABPβ1L (β1L), a tetramer-forming isoform of GABP that is dispensable for normal development, results in TERT silencing in a TERT promoter mutation-dependent manner. Reducing TERT expression by disrupting β1L culminates in telomere loss and cell death exclusively in TERT promoter mutant cells. Orthotopic xenografting of β1L-reduced, TERT promoter mutant glioblastoma cells rendered lower tumor burden and longer overall survival in mice. These results highlight the critical role of GABPβ1L in enabling immortality in TERT promoter mutant glioblastoma.This work was supported by a generous gift from the Dabbiere family (J.F.C.), the Hana Jabsheh Research Initiative (J.F.C.), NIH grant NCI P50CA097257 (J.F.C. and J.A.D.), NCI P01CA118816-06 (J.F.C.), T32 GM008568 and T32 CA151022 (A.M.), and NCI R01CA163336 (J.S.S.), and the Sontag Foundation Distinguished Scientist Award (J.S.S.). C.F. is supported by a US NIH K99/R00 Pathway to Independence Award (K99GM118909) from the National Institute of General Medical Sciences. Additional support was provided by Fundação para a Ciência e Tecnologia SFRH/BD/88220/2012 (A.X.-M.) and IF/00601/2012 (B.M.C.). J.A.D. is an investigator of the Howard Hughes Medical Institute.info:eu-repo/semantics/publishedVersio

RNA-based recognition and targeting: sowing the seeds of specificity

RNA is involved in the regulation of multiple cellular processes, often by forming sequence-specific base pairs with cellular RNA or DNA targets that must be identified among the large number of nucleic acids in a cell. Several RNA-based regulatory systems in eukaryotes, bacteria and archaea, including microRNAs (miRNAs), small interfering RNAs (siRNAs), CRISPR RNAs (crRNAs) and small RNAs (sRNAs) that are dependent on the RNA chaperone protein Hfq, achieve specificity using similar strategies. Central to their function is the presentation of short 'seed sequences' within a ribonucleoprotein complex to facilitate the search for and recognition of targets

Methods to Crystallize RNA

Preparation of suitably large and wellâ ordered single crystals is usually the rateâ limiting step in the determination of the threeâ dimensional structure of RNAs and their complexes with proteins by Xâ ray crystallography. This unit discusses a variety of experimental considerations for obtaining crystals of RNAs and RNAâ protein complexes. Topics include design of crystallizable constructs, screening, and optimization of crystallization conditions.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/152900/1/cpnc0706.pd

Structural biology. Structures of the CRISPR-Cmr complex reveal mode of RNA target positioning

Adaptive immunity in bacteria involves RNA-guided surveillance complexes that use CRISPR (clustered regularly interspaced short palindromic repeats)-associated (Cas) proteins together with CRISPR RNAs (crRNAs) to target invasive nucleic acids for degradation. Whereas type I and type II CRISPR-Cas surveillance complexes target double-stranded DNA, type III complexes target single-stranded RNA. Near-atomic resolution cryo-electron microscopy reconstructions of native type III Cmr (CRISPR RAMP module) complexes in the absence and presence of target RNA reveal a helical protein arrangement that positions the crRNA for substrate binding. Thumblike ß hairpins intercalate between segments of duplexed crRNA:target RNA to facilitate cleavage of the target at 6-nucleotide intervals. The Cmr complex is architecturally similar to the type I CRISPR-Cascade complex, suggesting divergent evolution of these immune systems from a common ancestor

Structure and Activity of the RNA-Targeting Type III-B CRISPR-Cas Complex of Thermus thermophilus

The CRISPR-Cas system is a prokaryotic host defense system against genetic elements. The Type III-B CRISPR-Cas system of the bacterium Thermus thermophilus, the TtCmr complex, is composed of six different protein subunits (Cmr1-6) and one crRNA with a stoichiometry of Cmr112131445361:crRNA1. The TtCmr complex copurifies with crRNA species of 40 and 46 nt, originating from a distinct subset of CRISPR loci and spacers. The TtCmr complex cleaves the target RNA at multiple sites with 6 nt intervals via a 5' ruler mechanism. Electron microscopy revealed that the structure of TtCmr resembles a "sea worm" and is composed of a Cmr2-3 heterodimer "tail," a helical backbone of Cmr4 subunits capped by Cmr5 subunits, and a curled "head" containing Cmr1 and Cmr6. Despite having a backbone of only four Cmr4 subunits and being both longer and narrower, the overall architecture of TtCmr resembles that of Type I Cascade complexe

Structure and Activity of the RNA-Targeting Type III-B CRISPR-Cas Complex of Thermus thermophilus

The CRISPR-Cas system is a prokaryotic host defense system against genetic elements. The Type III-B CRISPR-Cas system of the bacterium Thermus thermophilus, the TtCmr complex, is composed of six different protein subunits (Cmr1-6) and one crRNA with a stoichiometry of Cmr112131445361:crRNA1. The TtCmr complex copurifies with crRNA species of 40 and 46 nt, originating from a distinct subset of CRISPR loci and spacers. The TtCmr complex cleaves the target RNA at multiple sites with 6 nt intervals via a 5' ruler mechanism. Electron microscopy revealed that the structure of TtCmr resembles a "sea worm" and is composed of a Cmr2-3 heterodimer "tail," a helical backbone of Cmr4 subunits capped by Cmr5 subunits, and a curled "head" containing Cmr1 and Cmr6. Despite having a backbone of only four Cmr4 subunits and being both longer and narrower, the overall architecture of TtCmr resembles that of Type I Cascade complexe

RNA-targeting by the Type III-A CRISPR-Cas complex of Thermus thermophilus

crRNAs from the Thermus thermophilus CRISPR-Cas Csm comple