Cytoplasmic binding partners of the Integrator endonuclease INTS11 and its paralog CPSF73 are required for their nuclear function.

INTS11 and CPSF73 are metal-dependent endonucleases for Integrator and pre-mRNA 3'-end processing, respectively. Here, we show that the INTS11 binding partner BRAT1/CG7044, a factor important for neuronal fitness, stabilizes INTS11 in the cytoplasm and is required for Integrator function in the nucleus. Loss of BRAT1 in neural organoids leads to transcriptomic disruption and precocious expression of neurogenesis-driving transcription factors.

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

The sequence-specific RNA-binding protein Pumilio (Pum) controls 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 cells in culture. We also use an improved RNA coimmunoprecipitation method to identify Pum-bound mRNAs in embryos.

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

The sequence-specific RNA-binding protein Pumilio controls development of ; 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 cells. We also used an improved RNA co-immunoprecipitation method to identify Pumilio bound mRNAs in embryos.

INTS13 variants causing a recessive developmental ciliopathy disrupt assembly of the Integrator complex.

Oral-facial-digital (OFD) syndromes are a heterogeneous group of congenital disorders characterized by malformations of the face and oral cavity, and digit anomalies. Mutations within 12 cilia-related genes have been identified that cause several types of OFD, suggesting that OFDs constitute a subgroup of developmental ciliopathies. Through homozygosity mapping and exome sequencing of two families with variable OFD type 2, we identified distinct germline variants in INTS13, a subunit of the Integrator complex.

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.

Sine oculis homeobox homolog 1 plays a critical role in pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a fatal disease with limited treatment options. The role of the developmental transcription factor Sine oculis homeobox homolog 1 (SIX1) in the pathophysiology of lung fibrosis is not known. IPF lung tissue samples and IPF-derived alveolar type II cells (AT2) showed a significant increase in SIX1 mRNA and protein levels, and the SIX1 transcriptional coactivators EYA1 and EYA2 were elevated.

Generation and characterization of human Fetal membrane and Decidual cell lines for reproductive biology experiments†.

Human fetal membrane and maternal decidua parietalis form one of the major feto-maternal interfaces during pregnancy. Studies on this feto-maternal interface is limited as several investigators have limited access to the placenta, and experience difficulties to isolate and maintain primary cells. Many cell lines that are currently available do not have the characteristics or properties of their primary cells of origin.

RBFOX2 is critical for maintaining alternative polyadenylation patterns and mitochondrial health in rat myoblasts.

RBFOX2, which has a well-established role in alternative splicing, is linked to heart diseases. However, it is unclear whether RBFOX2 has other roles in RNA processing that can influence gene expression in muscle cells, contributing to heart disease. Here, we employ both 3'-end and nanopore cDNA sequencing to reveal a previously unrecognized role for RBFOX2 in maintaining alternative polyadenylation (APA) signatures in myoblasts.

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.

An atlas of alternative polyadenylation quantitative trait loci contributing to complex trait and disease heritability.

Genome-wide association studies have identified thousands of noncoding variants associated with human traits and diseases. However, the functional interpretation of these variants is a major challenge. Here, we constructed a multi-tissue atlas of human 3'UTR alternative polyadenylation (APA) quantitative trait loci (3'aQTLs), containing approximately 0.

Human Pumilio proteins directly bind the CCR4-NOT deadenylase complex to regulate the transcriptome.

Pumilio paralogs, PUM1 and PUM2, are sequence-specific RNA-binding proteins that are essential for vertebrate development and neurological functions. PUM1&2 negatively regulate gene expression by accelerating degradation of specific mRNAs. Here, we determined the repression mechanism and impact of human PUM1&2 on the transcriptome.

Integrator Recruits Protein Phosphatase 2A to Prevent Pause Release and Facilitate Transcription Termination.

Efficient release of promoter-proximally paused RNA Pol II into productive elongation is essential for gene expression. Recently, we reported that the Integrator complex can bind paused RNA Pol II and drive premature transcription termination, potently attenuating the activity of target genes. Premature termination requires RNA cleavage by the endonuclease subunit of Integrator, but the roles of other Integrator subunits in gene regulation have yet to be elucidated.

The Integrator Complex Attenuates Promoter-Proximal Transcription at Protein-Coding Genes.

The transition of RNA polymerase II (Pol II) from initiation to productive elongation is a central, regulated step in metazoan gene expression. At many genes, Pol II pauses stably in early elongation, remaining engaged with the 25- to 60-nt-long nascent RNA for many minutes while awaiting signals for release into the gene body. However, 15%-20% of genes display highly unstable promoter Pol II, suggesting that paused polymerase might dissociate from template DNA at these promoters and release a short, non-productive mRNA.

The Integrator complex cleaves nascent mRNAs to attenuate transcription.

Cellular homeostasis requires transcriptional outputs to be coordinated, and many events post-transcription initiation can dictate the levels and functions of mature transcripts. To systematically identify regulators of inducible gene expression, we performed high-throughput RNAi screening of the Metallothionein A (MtnA) promoter. This revealed that the Integrator complex, which has a well-established role in 3' end processing of small nuclear RNAs (snRNAs), attenuates MtnA transcription during copper stress.

Development of Poly(A)-ClickSeq as a tool enabling simultaneous genome-wide poly(A)-site identification and differential expression analysis.

The use of RNA-seq as a generalized tool to measure the differential expression of genes has essentially replaced the use of the microarray. Despite the acknowledged technical advantages to this approach, RNA-seq library preparation remains mostly conducted by core facilities rather than in the laboratory due to the infrastructure, expertise and time required per sample. We have recently described two 'click-chemistry' based library construction methods termed ClickSeq and Poly(A)-ClickSeq (PAC-seq) as alternatives to conventional RNA-seq that are both cost effective and rely on straightforward reagents readily available to most labs.