Biology subjects

Connacher, R. P.

Publications and source records attributed to Connacher, R. P..

3 recordsLinked to original sources

bioRxiv2026

Global Regulatory Functions of the Human CCR4-NOT Complex in mRNA Deadenylation and Decay

Shortening of the poly(A) tail initiates mRNA decay, and the human CCR4-NOT is the principal cytoplasmic deadenylase. Although its subunits have been defined, their functions have not been comprehensively evaluated in cells. We depleted each CCR4-NOT subunit and measured transcriptome-wide effects. CCR4-NOT regulated transcripts from thousands of genes, many of which associated with the complex in cells. Distinct groups of transcripts responded according to the modular organization of the complex, while subsets exhibited unique responses to specific subunits. Poly(A) tail analysis revealed that the CNOT7 and CNOT8 deadenylases are the predominant regulators of poly(A) tail length in these cells. CCR4-NOT targets were enriched in regulatory pathways including cell proliferation, apoptosis, stress responses, and cancer-related processes. Bioinformatic analysis identified sequence determinants and candidate recruitment factors associated with CCR4-NOT regulation. The results define the breadth and specificity of CCR4-NOT-mediated mRNA decay and provide a resource for dissecting regulatory mechanisms and biological roles.

biochemistry↗

bioRxiv2026

RNA-binding is the essential biological function of the Drosophila protein Brat

Brain tumor (Brat) is a Drosophila TRIM-NHL protein required for embryogenesis and neural stem cell differentiation. Although structural and biochemical studies established that the Brat NHL domain specifically binds RNA, the in vivo requirement for this activity has not been directly tested. Here, we used structure-guided mutagenesis and genome engineering to determine whether RNA recognition is essential for Brat function during development. The direct interaction between Brats NHL domain and RNA containing Brat Binding Sites (BBS) can be abolished by alanine substitution of three separate residues on the NHL surface. We introduced these point mutations into the endogenous brat locus by CRISPR-mediated Scarless Gene Editing to generate three independent RNA-binding defective mutant (RBDmt) alleles. Complementation tests demonstrated that each allele behaves as a strong loss-of-function mutation: homozygotes and hemizygotes are inviable, and RBDmt alleles fail to complement classical brat null and hypomorphic alleles. Lethal phase analysis revealed death predominantly during late larval and pupal stages, consistent with known brat alleles. Consistent with the namesake brat phenotype, RBDmt larval brains exhibited widespread expression of neuroblast markers and a marked reduction of neuronal differentiation. In embryos, these alleles failed to complement female sterile brat alleles and recapitulated characteristic abdominal segmentation defects. Finally, RT-qPCR showed increased expression of endogenous Brat target mRNAs in mutant larvae, consistent with loss of Brat-mediated repression. Together, these results demonstrate that direct RNA binding is the essential molecular activity of Brat and that post-transcriptional regulation of Brat target mRNAs underlies its critical roles across development.

molecular biology↗

bioRxiv2023

Chemi-Northern: a versatile chemiluminescent northern blot method for analysis and quantitation of RNA molecules.

This report describes a chemiluminescence-based detection method for RNAs on northern blots, designated Chemi-Northern. This approach builds on the simplicity and versatility of northern blotting, while dispensing of the need for expensive and cumbersome radioactivity. RNAs are first separated on denaturing gel electrophoresis, transferred to a nylon membrane, and then hybridized to a biotinylated RNA or DNA antisense probe. Streptavidin conjugated with horseradish peroxidase and enhanced chemiluminescence substrate are then used to detect the probe bound to the target RNA. Our results demonstrate the versatility of this method in detecting natural and engineered RNAs expressed in cells, including messenger and noncoding RNAs. We show that Chemi-Northern detection is sensitive and fast, detecting attomole amounts of RNA in as little as 1 second, with high signal intensity and low background. The dynamic response displays excellent linearity. Using Chemi-Northern, we measure the significant, reproducible reduction of mRNA levels by human sequence-specific RNA-binding proteins, PUM1 and PUM2. Additionally, we measure the interaction of endogenous poly(A) binding protein, PABPC1, with poly-adenylated mRNA. Thus, the Chemi-Northern method provides a versatile, simple, cost-effective method to enable researchers to detect and measure changes in RNA expression, processing, binding, and decay of RNAs.

molecular biology↗