Enhancing Transcriptome Mapping with Rapid PRO-seq Profiling of Nascent RNA.

Precision nuclear run-on (PRO) sequencing (PRO-seq) is a powerful technique for mapping polymerase active sites with nucleotide resolution and measuring newly synthesized transcripts at both promoters and enhancer elements. The current PRO-seq protocol is time-intensive, technically challenging, and requires a large amount of starting material. To overcome these limitations, we developed rapid PRO-seq (rPRO-seq) which utilizes pre-adenylated single-stranded DNAs (AppDNA), a dimer blocking oligonucleotide (DBO), on-bead 5' RNA end repair, and column-based purification. These modifications enabled efficient transcriptome mapping within a single day (∼12 hours) increasing ligation efficiency, abolished adapter dimers, and reduced sample loss and RNA degradation. We demonstrate the reproducibility of rPRO-seq in measuring polymerases at promoters, gene bodies, and enhancers as compared to original PRO-seq protocols. Additionally, rPRO-seq is scalable, allowing for transcriptome mapping with as little as 25,000 cells. We apply rPRO-seq to study the role of Integrator in mouse hematopoietic stem and progenitor cell (mHSPC) homeostasis, identifying Ints11 as an essential component of transcriptional regulation and RNA processing in mHSPC homeostasis. Overall, rPRO-seq represents a significant advance in the field of nascent transcript analyses and will be a valuable tool for generating patient-specific genome-wide transcription profiles with minimal sample requirements.

The Integrator complex regulates microRNA abundance through RISC loading.

MicroRNA (miRNA) homeostasis is crucial for the posttranscriptional regulation of their target genes during development and in disease states. miRNAs are derived from primary transcripts and are processed from a hairpin precursor intermediary to a mature 22-nucleotide duplex RNA. Loading of the duplex into the Argonaute (AGO) protein family is pivotal to miRNA abundance and its posttranscriptional function. The Integrator complex plays a key role in protein coding and noncoding RNA maturation, RNA polymerase II pause-release, and premature transcriptional termination. Here, we report that loss of Integrator results in global destabilization of mature miRNAs. Enhanced ultraviolet cross-linking and immunoprecipitation of Integrator uncovered an association with duplex miRNAs before their loading onto AGOs. Tracing miRNA fate from biogenesis to stabilization by incorporating 4-thiouridine in nascent transcripts pinpointed a critical role for Integrator in miRNA assembly into AGOs.

Membrane Domain Localization and Interaction of the Prion-Family Proteins, Prion and Shadoo with Calnexin.

The cellular prion protein (PrPC) is renowned for its infectious conformational isoform PrPSc, capable of templating subsequent conversions of healthy PrPCs and thus triggering the group of incurable diseases known as transmissible spongiform encephalopathies. Besides this mechanism not being fully uncovered, the protein's physiological role is also elusive. PrPC and its newest, less understood paralog Shadoo are glycosylphosphatidylinositol-anchored proteins highly expressed in the central nervous system. While they share some attributes and neuroprotective actions, opposing roles have also been reported for the two; however, the amount of data about their exact functions is lacking. Protein-protein interactions and membrane microdomain localizations are key determinants of protein function. Accurate identification of these functions for a membrane protein, however, can become biased due to interactions occurring during sample processing. To avoid such artifacts, we apply a non-detergent-based membrane-fractionation approach to study the prion protein and Shadoo. We show that the two proteins occupy similarly raft and non-raft membrane fractions when expressed in N2a cells and that both proteins pull down the chaperone calnexin in both rafts and non-rafts. These indicate their possible binding to calnexin in both types of membrane domains, which might be a necessary requisite to aid the inherently unstable native conformation during their lifetime.

Integrator enforces the fidelity of transcriptional termination at protein-coding genes.

Integrator regulates the 3'-end processing and termination of multiple classes of noncoding RNAs. Depletion of INTS11, the catalytic subunit of Integrator, or ectopic expression of its catalytic dead enzyme impairs the 3'-end processing and termination of a set of protein-coding transcripts termed Integrator-regulated termination (IRT) genes. This defect is manifested by increased RNA polymerase II (RNAPII) readthrough and occupancy of serine-2 phosphorylated RNAPII, de novo trimethylation of lysine-36 on histone H3, and a compensatory elevation of the cleavage and polyadenylation (CPA) complex beyond the canonical polyadenylation sites. 3' RNA sequencing reveals that proximal polyadenylation site usage relies on the endonuclease activity of INTS11. The DNA sequence encompassing the transcription end sites of IRT genes features downstream polyadenylation motifs and an enrichment of GC content that permits the formation of secondary structures within the 3'UTR. Together, this study identifies a subset of protein-coding transcripts whose 3' end processing requires the Integrator complex.

RAC1 plays an essential role in estrogen receptor alpha function in breast cancer cells.

The activity of Rho family GTPase protein, RAC1, which plays important normal physiological functions, is dysregulated in multiple cancers. RAC1 is expressed in both estrogen receptor alpha (ER)-positive and ER-negative breast cancer (BC) cells. However, ER-positive BC is more sensitive to RAC1 inhibition. We have determined that reducing RAC1 activity, using siRNA or EHT 1864 (a small molecule Rac inhibitor), leads to rapid ER protein degradation. RAC1 interacts with ER within the ER complex and RAC1 localizes to chromatin binding sites for ER upon estrogen treatment. RAC1 activity is important for RNA Pol II function at both promoters and enhancers of ER target genes and ER-regulated gene transcription is blocked by EHT 1864, in a dose-dependent manner. Having identified that RAC1 is an essential ER cofactor for ER protein stability and ER transcriptional activity, we report that RAC1 inhibition could be an effective therapeutic approach for ER-positive BC.

The Human Integrator Complex Facilitates Transcriptional Elongation by Endonucleolytic Cleavage of Nascent Transcripts.

Transcription by RNA polymerase II (RNAPII) is pervasive in the human genome. However, the mechanisms controlling transcription at promoters and enhancers remain enigmatic. Here, we demonstrate that Integrator subunit 11 (INTS11), the catalytic subunit of the Integrator complex, regulates transcription at these loci through its endonuclease activity. Promoters of genes require INTS11 to cleave nascent transcripts associated with paused RNAPII and induce their premature termination in the proximity of the +1 nucleosome. The turnover of RNAPII permits the subsequent recruitment of an elongation-competent RNAPII complex, leading to productive elongation. In contrast, enhancers require INTS11 catalysis not to evict paused RNAPII but rather to terminate enhancer RNA transcription beyond the +1 nucleosome. These findings are supported by the differential occupancy of negative elongation factor (NELF), SPT5, and tyrosine-1-phosphorylated RNAPII. This study elucidates the role of Integrator in mediating transcriptional elongation at human promoters through the endonucleolytic cleavage of nascent transcripts and the dynamic turnover of RNAPII.