Regulation of the Drosophila transcriptome by Pumilio and the CCR4-NOT deadenylase complex.

The sequence-specific RNA-binding protein Pumilio (Pum) controls Drosophila development; however, the network of mRNAs that it regulates remains incompletely characterized. In this study, we use knockdown and knockout approaches coupled with RNA-seq to measure the impact of Pum on the transcriptome of Drosophila cells in culture. We also use an improved RNA coimmunoprecipitation method to identify Pum-bound mRNAs in Drosophila embryos. Integration of these data sets with the locations of Pum-binding motifs across the transcriptome reveals novel direct Pum target genes involved in neural, muscle, wing, and germ cell development and in cellular proliferation. These genes include components of Wnt, TGF-ß, MAPK/ERK, and Notch signaling pathways, DNA replication, and lipid metabolism. We identify the mRNAs regulated by the CCR4-NOT deadenylase complex, a key factor in Pum-mediated repression, and observe concordant regulation of Pum:CCR4-NOT target mRNAs. Computational modeling reveals that Pum binding, binding site number, clustering, and sequence context are important determinants of regulation. In contrast, we show that the responses of direct mRNA targets to Pum-mediated repression are not influenced by the content of optimal synonymous codons. Moreover, contrary to a prevailing model, we do not detect a role for CCR4-NOT in the degradation of mRNAs with low codon optimality. Together, the results of this work provide new insights into the Pum regulatory network and mechanisms and the parameters that influence the efficacy of Pum-mediated regulation.

Regulation of the Drosophila transcriptome by Pumilio and CCR4-NOT deadenylase.

The sequence-specific RNA-binding protein Pumilio controls development of Drosophila; however, the network of mRNAs that it regulates remains incompletely characterized. In this study, we utilize knockdown and knockout approaches coupled with RNA-Seq to measure the impact of Pumilio on the transcriptome of Drosophila cells. We also used an improved RNA co-immunoprecipitation method to identify Pumilio bound mRNAs in Drosophila embryos. Integration of these datasets with the content of Pumilio binding motifs across the transcriptome revealed novel direct Pumilio target genes involved in neural, muscle, wing, and germ cell development, and cellular proliferation. These genes include components of Wnt, TGF-beta, MAPK/ERK, and Notch signaling pathways, DNA replication, and lipid metabolism. Additionally, we identified the mRNAs regulated by the CCR4-NOT deadenylase complex, a key factor in Pumilio-mediated repression, and observed concordant regulation of Pumilio:CCR4-NOT target mRNAs. Computational modeling revealed that Pumilio binding, binding site number, density, and sequence context are important determinants of regulation. Moreover, the content of optimal synonymous codons in target mRNAs exhibits a striking functional relationship to Pumilio and CCR4-NOT regulation, indicating that the inherent translation efficiency and stability of the mRNA modulates their response to these trans-acting regulatory factors. Together, the results of this work provide new insights into the Pumilio regulatory network and mechanisms, and the parameters that influence the efficacy of Pumilio-mediated regulation.

Inositol hexakisphosphate is required for Integrator function.

Integrator is a multi-subunit protein complex associated with RNA polymerase II (Pol II), with critical roles in noncoding RNA 3'-end processing and transcription attenuation of a broad collection of mRNAs. IntS11 is the endonuclease for RNA cleavage, as a part of the IntS4-IntS9-IntS11 Integrator cleavage module (ICM). Here we report a cryo-EM structure of the Drosophila ICM, at 2.74 Å resolution, revealing stable association of an inositol hexakisphosphate (IP6) molecule. The IP6 binding site is located in a highly electropositive pocket at an interface among all three subunits of ICM, 55 Å away from the IntS11 active site and generally conserved in other ICMs. We also confirmed IP6 association with the same site in human ICM. IP6 binding is not detected in ICM samples harboring mutations in this binding site. Such mutations or disruption of IP6 biosynthesis significantly reduced Integrator function in snRNA 3'-end processing and mRNA transcription attenuation. Our structural and functional studies reveal that IP6 is required for Integrator function in Drosophila, humans, and likely other organisms.

Application and design considerations for 3'-end sequencing using click-chemistry.

Over the past 15 years, investigations into alternative polyadenylation (APA) and its function in cellular physiology and pathology have greatly expanded due to the emergent appreciation of its key role in driving transcriptomic diversity. This growth has necessitated the development of new technologies capable of monitoring cleavage and polyadenylation events genome-wide. Advancements in approaches include both the creation of computational tools to re-analyze RNA-seq to identify APA events as well as targeted sequencing approaches customized to focus on the 3'-end of mRNA. Here we describe a streamlined protocol for polyA-Click-seq (PAC-seq), which utilizes click-chemistry to create mRNA 3'-ends sequencing libraries. Importantly, we offer additional considerations not present in our previous protocols including the use of spike-ins, unique molecular identifier primers, and guidance for appropriate depth of PAC-seq. In conjunction with the companion chapter on PolyA-miner (Yalamanchili et al., 2021) to computationally analyze PAC-seq data, we provide a complete experimental pipeline to analyze mRNA 3'-end usage in eukaryotic cells.

A computational pipeline to infer alternative poly-adenylation from 3' sequencing data.

An increasing number of investigations have established alternative polyadenylation (APA) as a key mechanism of gene regulation through altering the length of 3' untranslated region (UTR) and generating distinct mRNA termini. Further, appreciation for the significance of APA in disease contexts propelled the development of several 3' sequencing techniques. While these RNA sequencing technologies have advanced APA analysis, the intrinsic limitation of 3' read coverage and lack of appropriate computational tools constrain precise mapping and quantification of polyadenylation sites. Notably, Poly(A)-ClickSeq (PAC-seq) overcomes limiting factors such as poly(A) enrichment and 3' linker ligation steps using click-chemistry. Here we provide an updated PolyA-miner protocol, a computational approach to analyze PAC-seq or other 3'-Seq datasets. As a key practical constraint, we also provide a detailed account on the impact of sequencing depth on the number of detected polyadenylation sites and APA changes. This protocol is also updated to handle unique molecular identifiers used to address PCR duplication potentially observed in PAC-seq.