Molecular basis for nuclear accumulation and targeting of the inhibitor of apoptosis BIRC2.

The inhibitor of apoptosis protein BIRC2 regulates fundamental cell death and survival signaling pathways. Here we show that BIRC2 accumulates in the nucleus via binding of its second and third BIR domains, BIRC2BIR2 and BIRC2BIR3, to the histone H3 tail and report the structure of the BIRC2BIR3-H3 complex. RNA-seq analysis reveals that the genes involved in interferon and defense response signaling and cell-cycle regulation are most affected by depletion of BIRC2. Overexpression of BIRC2 delays DNA damage repair and recovery of the cell-cycle progression. We describe the structural mechanism for targeting of BIRC2BIR3 by a potent but biochemically uncharacterized small molecule inhibitor LCL161 and demonstrate that LCL161 disrupts the association of endogenous BIRC2 with H3 and stimulates cell death in cancer cells. We further show that LCL161 mediates degradation of BIRC2 in human immunodeficiency virus type 1-infected human CD4+ T cells. Our findings provide mechanistic insights into the nuclear accumulation of and blocking BIRC2.

cIAP1/TRAF2 interplay promotes tumor growth through the activation of STAT3.

Cellular inhibitor of apoptosis-1 (cIAP1) is a signaling regulator with oncogenic properties. It is involved in the regulation of signaling pathways controlling inflammation, cell survival, proliferation, differentiation and motility. It is recruited into membrane-receptor-associated signaling complexes thanks to the molecular adaptor TRAF2. However, the cIAP1/TRAF2 complex exists, independently of receptor engagement, in several subcellular compartments. The present work strengthens the importance of TRAF2 in the oncogenic properties of cIAP1. cIAPs-deficient mouse embryonic fibroblasts (MEFs) were transformed using the HRas-V12 oncogene. Re-expression of cIAP1 enhanced tumor growth in a nude mice xenograft model, and promoted lung tumor nodes formation. Deletion or mutation of the TRAF2-binding site completely abolished the oncogenic properties of cIAP1. Further, cIAP1 mediated the clustering of TRAF2, which was sufficient to stimulate tumor growth. Our TRAF2 interactome analysis showed that cIAP1 was critical for TRAF2 to bind to its protein partners. Thus, cIAP1 and TRAF2 would be two essential subunits of a signaling complex promoting a pro-tumoral signal. cIAP1/TRAF2 promoted the activation of the canonical NF-κB and ERK1/2 signaling pathways. NF-κB-dependent production of IL-6 triggered the activation of the JAK/STAT3 axis in an autocrine manner. Inhibition or downregulation of STAT3 specifically compromised the growth of cIAP1-restored MEFs but not that of MEFs expressing a cIAP1-mutant and treating mice with the STAT3 inhibitor niclosamide completely abrogated cIAP1/TRAF2-mediated tumor growth. Altogether, we demonstrate that cIAP1/TRAF2 binding is essential to promote tumor growth via the activation of the JAK/STAT3 signaling pathway.

Downregulation of Elovl5 promotes breast cancer metastasis through a lipid-droplet accumulation-mediated induction of TGF-ß receptors.

Metastatic breast cancer cannot be cured, and alteration of fatty acid metabolism contributes to tumor progression and metastasis. Here, we were interested in the elongation of very long-chain fatty acids protein 5 (Elovl5) in breast cancer. We observed that breast cancer tumors had a lower expression of Elovl5 than normal breast tissues. Furthermore, low expression of Elovl5 is associated with a worse prognosis in ER+ breast cancer patients. In accordance with this finding, decrease of Elovl5 expression was more pronounced in ER+ breast tumors from patients with metastases in lymph nodes. Although downregulation of Elovl5 expression limited breast cancer cell proliferation and cancer progression, suppression of Elovl5 promoted EMT, cell invasion and lung metastases in murine breast cancer models. The loss of Elovl5 expression induced upregulation of TGF-ß receptors mediated by a lipid-droplet accumulation-dependent Smad2 acetylation. As expected, inhibition of TGF-ß receptors restored proliferation and dampened invasion in low Elovl5 expressing cancer cells. Interestingly, the abolition of lipid-droplet formation by inhibition of diacylglycerol acyltransferase activity reversed induction of TGF-ß receptors, cell invasion, and lung metastasis triggered by Elovl5 knockdown. Altogether, we showed that Elovl5 is involved in metastasis through lipid droplets-regulated TGF-ß receptor expression and is a predictive biomarker of metastatic ER+ breast cancer.

Heat shock proteins and exosomes in cancer theranostics.

Heat shock proteins (HSPs) are a superfamily of molecular chaperones that were discovered through their ability to be induced by different stresses including heat shock. Other than their function as chaperones in proteins homeostasis, HSPs have been shown to inhibit different forms of cell death and to participate in cell proliferation and differentiation processes. Because cancer cells have to rewire their metabolism, they require a high amount of these stress-inducible chaperones for their survival. Therefore, HSPs are unusually abundant in cancer cells where they have oncogene-like functions. In cancer, HSPs have been involved in the regulation of apoptosis, immune responses, angiogenesis, metastasis and treatment resistance. Recently, HSPs have been shown to be secreted through exosomes by cancer cells. These tumor-derived exosomes can be used as circulating markers: HSP-exosomes have been reported as biomarkers of cancer dissemination, response to therapy and/or patient outcome. A new range of functions, mostly in modulation of anticancer immune responses, have been described for these extracellular HSPs. In this review, we will describe those recently reported functions of HSP-exosomes that makes them both targets for anticancer therapeutics and biomarkers for the monitoring of the disease. We will also discuss their emerging interest in cancer vaccines.

Endoplasmic Reticulum Chaperones in Viral Infection: Therapeutic Perspectives.

Viruses are intracellular parasites that subvert the functions of their host cells to accomplish their infection cycle. The endoplasmic reticulum (ER)-residing chaperone proteins are central for the achievement of different steps of the viral cycle, from entry and replication to assembly and exit. The most abundant ER chaperones are GRP78 (78-kDa glucose-regulated protein), GRP94 (94-kDa glucose-regulated protein), the carbohydrate or lectin-like chaperones calnexin (CNX) and calreticulin (CRT), the protein disulfide isomerases (PDIs), and the DNAJ chaperones. This review will focus on the pleiotropic roles of ER chaperones during viral infection. We will cover their essential role in the folding and quality control of viral proteins, notably viral glycoproteins which play a major role in host cell infection. We will also describe how viruses co-opt ER chaperones at various steps of their infectious cycle but also in order to evade immune responses and avoid apoptosis. Finally, we will discuss the different molecules targeting these chaperones and the perspectives in the development of broad-spectrum antiviral drugs.

Tumor-Derived Exosomes: Hidden Players in PD-1/PD-L1 Resistance.

Recently, immunotherapy has garnered increasing importance in cancer therapy, leading to substantial improvements in patient care and survival. By blocking the immune checkpoints-protein regulators of the immune system-immunotherapy prevents immune tolerance toward tumors and reactivates the immune system, prompting it to fight cancer cell growth and diffusion. A widespread strategy for this is the blockade of the interaction between PD-L1 and PD-1. However, while patients generally respond well to immunotherapy, a certain proportion of patients present tumors that resist these treatments. This portion can be very high in some cancers and hinders cancer curability. For this reason, current efforts are focusing on combining PD-1/PD-L1 immunotherapy with the targeting of other immune checkpoints to counter resistance and achieve better results. Exosomes, small vesicles secreted by almost any cell, including tumor cells, have proven to be key actors in this resistance. The exosomes released by tumor cells spread the immune-suppressive properties of the tumor throughout the tumor microenvironment and participate in establishing metastatic niches. In this review, we will describe immune checkpoints and immune modulators whose presence in tumor-derived exosomes (TEXs) has been established. We will focus on the most promising proteins under scrutiny for use in combination with PD-1 blockade therapy in a clinical setting, such as PD-L1, CTLA-4, TIM-3, CD73/39, LAG-3, and TIGIT. We will explore the immunosuppressive impact of these exosomal proteins on a variety of immune cells. Finally, we will discuss how they can change the game in immunotherapy and guide therapeutic decisions, as well as the current limits of this approach. Depending on the viewpoint, these exosomal proteins may either provide key missing information on tumor growth and resistance mechanisms or they may be the next big challenge to overcome in improving cancer treatment.

Lactobacillus stress protein GroEL prevents colonic inflammation.

BACKGROUND: We previously showed that supernatants of Lactobacillus biofilms induced an anti-inflammatory response by affecting the secretion of macrophage-derived cytokines, which was abrogated upon immunodepletion of the stress protein GroEL. METHODS: We purified GroEL from L. reuteri and analysed its anti-inflammatory properties in vitro in human macrophages isolated from buffy coats, ex vivo in explants from human biopsies and in vivo in a mouse model of DSS induced intestinal inflammation. As a control, we used GroEL purified (LPS-free) from E. coli. RESULTS: We found that L. reuteri GroEL (but not E. coli GroEL) inhibited pro-inflammatory M1-like macrophages markers, and favored M2-like markers. Consequently, L. reuteri GroEL inhibited pro-inflammatory cytokines (TNFα, IL-1ß, IFNγ) while favouring an anti-inflammatory secretome. In colon tissues from human biopsies, L. reuteri GroEL was also able to decrease markers of inflammation and apoptosis (caspase 3) induced by LPS. In mice, we found that rectal administration of L. reuteri GroEL (but not E. coli GroEL) inhibited all signs of haemorrhagic colitis induced by DSS including intestinal mucosa degradation, rectal bleeding and weight loss. It also decreased intestinal production of inflammatory cytokines (such as IFNγ) while increasing anti-inflammatory IL-10 and IL-13. These effects were suppressed when animals were immunodepleted in macrophages. From a mechanistic point of view, the effect of L. reuteri GroEL seemed to involve TLR4, since it was lost in TRL4-/- mice, and the activation of a non-canonical TLR4 pathway. CONCLUSIONS: L. reuteri GroEL, by affecting macrophage inflammatory features, deserves to be explored as an alternative to probiotics.

XPO1 regulates erythroid differentiation and is a new target for the treatment of ß-thalassemia.

ß-thalassemia major (ß-TM) is an inherited hemoglobinopathy caused by a quantitative defect in the synthesis of ß-globin chains of hemoglobin, leading to the accumulation of free a-globin chains that aggregate and cause ineffective erythropoiesis. We have previously demonstrated that terminal erythroid maturation requires a transient activation of caspase-3 and that the chaperone Heat Shock Protein 70 (HSP70) accumulates in the nucleus to protect GATA-1 transcription factor from caspase-3 cleavage. This nuclear accumulation of HSP70 is inhibited in human ß-TM erythroblasts due to HSP70 sequestration in the cytoplasm by free a-globin chains, resulting in maturation arrest and apoptosis. Likewise, terminal maturation can be restored by transduction of a nuclear-targeted HSP70 mutant. Here we demonstrate that in normal erythroid progenitors, HSP70 localization is regulated by the exportin-1 (XPO1), and that treatment of ß-thalassemic erythroblasts with an XPO1 inhibitor increased the amount of nuclear HSP70, rescued GATA-1 expression and improved terminal differentiation, thus representing a new therapeutic option to ameliorate ineffective erythropoiesis of ß-TM.

Heat-shock proteins: chaperoning DNA repair.

Cells are repeatedly exposed to environmental or endogenous stresses that can alter normal cell behavior and increase cell vulnerability. In order to ensure tissue integrity and function, cells cope with cellular injuries by adapting their metabolism, protecting essential intracellular constituents, inhibiting cell death signaling pathways and activating those devoted to damage repair. The molecular chaperones of the heat-shock protein (HSP) family are critical effectors of this adaptive response. They protect intracellular proteins from misfolding or aggregation, inhibit cell death signaling cascades and preserve the intracellular signaling pathways that are essential for cell survival. Most HSPs are rapidly overexpressed in response to cellular injuries including genotoxic stress. DNA damage can dramatically alter cell behavior and contribute to a number of diseases including developmental defects, neurodegenerative disorders, and cancer. Thus, the ability of cells to repair DNA damage is essential for preserving cell integrity. DNA damage activates a coordinated response that includes detecting DNA lesions before their transmission to daughter cells, blocking cell cycle progression and DNA replication and repairing the damage. Although the role of HSPs in proteins homeostasis and cell death, especially apoptosis has been widely reported, much less is known about their function in DNA repair. This review aims to present the role of HSPs in DNA repair signaling pathways.

HSP110 translocates to the nucleus upon genotoxic chemotherapy and promotes DNA repair in colorectal cancer cells.

A multicenter clinical study demonstrated the presence of a loss-of-function HSP110 mutation in about 15% of colorectal cancers, which resulted from an alternative splicing and was produced at the detriment of wild-type HSP110. Patients expressing low levels of wild-type HSP110 had excellent outcomes (i.e. response to an oxaliplatin-based chemotherapy). Here, we show in vitro, in vivo, and in patients' biopsies that HSP110 co-localizes with DNA damage (γ-H2AX). In colorectal cancer cells, HSP110 translocates into the nucleus upon treatment with genotoxic chemotherapy such as oxaliplatin. Furthermore, we show that HSP110 interacts with the Ku70/Ku80 heterodimer, an essential element of the non-homologous end joining (NHEJ) repair machinery. We also demonstrate by evaluating the resolved 53BP1 foci that depletion in HSP110 impairs repair steps of the NHEJ pathway, which is associated with an increase in DNA double-strand breaks and in the cells' sensitivity to oxaliplatin. HSP110-depleted cells sensitization to oxaliplatin-induced DNA damage is abolished upon re-expression of HSP110. Confirming a role for HSP110 in DNA non-homologous repair, SCR7 and NU7026, two inhibitors of the NHEJ pathway, circumvents HSP110-induced resistance to chemotherapy. In conclusion, HSP110 through its interaction with the Ku70/80 heterodimer may participate in DNA repair, thereby inducing a protection against genotoxic therapy.

HSP27 is a partner of JAK2-STAT5 and a potential therapeutic target in myelofibrosis.

Heat shock protein 27 (HSP27/HSPB1) is a stress-inducible chaperone that facilitates cancer development by its proliferative and anti-apoptotic functions. The OGX-427 antisense oligonucleotide against HSP27 has been reported to be beneficial against idiopathic pulmonary fibrosis. Here we show that OGX-427 is effective in two murine models of thrombopoietin- and JAKV617F-induced myelofibrosis. OGX-427 limits disease progression and is associated with a reduction in spleen weight, in megakaryocyte expansion and, for the JAKV617F model, in fibrosis. HSP27 regulates the proliferation of JAK2V617F-positive cells and interacts directly with JAK2/STAT5. We also show that its expression is increased in both CD34+ circulating progenitors and in the serum of patients with JAK2-dependent myeloproliferative neoplasms with fibrosis. Our data suggest that HSP27 plays a key role in the pathophysiology of myelofibrosis and represents a new potential therapeutic target for patients with myeloproliferative neoplasms.

N-glycosylation of mouse TRAIL-R and human TRAIL-R1 enhances TRAIL-induced death.

APO2L/TRAIL (TNF-related apoptosis-inducing ligand) induces death of tumor cells through two agonist receptors, TRAIL-R1 and TRAIL-R2. We demonstrate here that N-linked glycosylation (N-glyc) plays also an important regulatory role for TRAIL-R1-mediated and mouse TRAIL receptor (mTRAIL-R)-mediated apoptosis, but not for TRAIL-R2, which is devoid of N-glycans. Cells expressing N-glyc-defective mutants of TRAIL-R1 and mouse TRAIL-R were less sensitive to TRAIL than their wild-type counterparts. Defective apoptotic signaling by N-glyc-deficient TRAIL receptors was associated with lower TRAIL receptor aggregation and reduced DISC formation, but not with reduced TRAIL-binding affinity. Our results also indicate that TRAIL receptor N-glyc impacts immune evasion strategies. The cytomegalovirus (CMV) UL141 protein, which restricts cell-surface expression of human TRAIL death receptors, binds with significant higher affinity TRAIL-R1 lacking N-glyc, suggesting that this sugar modification may have evolved as a counterstrategy to prevent receptor inhibition by UL141. Altogether our findings demonstrate that N-glyc of TRAIL-R1 promotes TRAIL signaling and restricts virus-mediated inhibition.

Pleural inhibition of the caspase-1/IL-1ß pathway diminishes profibrotic lung toxicity of bleomycin.

BACKGROUND: Idiopathic and toxic pulmonary fibrosis are severe diseases starting classically in the subpleural area of the lung. It has recently been suggested that pleural mesothelial cells acquire a myofibroblast phenotype under fibrotic conditions induced by TGF-ß1 or bleomycin. The importance and role of inflammation in fibrogenesis are still controversial. In this work, we explored the role of IL-1ß/caspase-1 signaling in bleomycin lung toxicity and in pleural mesothelial cell transformation. METHODS: C57BL/6 mice were intravenously injected with either bleomycin or nigericin or NaCl as control. In vitro, the Met5A cell line was used as a model of human pleural mesothelial cells. RESULTS: Intravenous injections of bleomycin induced lung fibrosis with histologically-proven peripheral distribution, collagen accumulation in the pleural and subpleural area, and overexpression of markers of myofibroblast transformation of pleural cells which migrated into the lung. These events were associated with an inflammatory process with an increase in neutrophil recruitment in pleural lavage fluid and increased caspase-1 activity. TGF-ß1 was also overexpressed in pleural lavage fluid and was produced by pleural cells following intravenous bleomycin. In this model, local pleural inhibition of IL-1ß with the IL-1ß inhibitor anakinra diminished TGF-ß1 and collagen accumulation. In vitro, caspase-1 inhibition interfered with Met5A cell transformation into the myofibroblast-like phenotype induced by bleomycin or TGF-ß1. Moreover, nigericin, a caspase-1 activator, triggered transformation of Met5A cells and its intra-pleural delivery induced fibrogenesis in mice. CONCLUSIONS: We demonstrated, after intravenous bleomycin injection in mice, the role of the pleura and highlighted the key role of IL-1ß/caspase-1 axis in this fibrogenesis process.

Extracellular HSP110 skews macrophage polarization in colorectal cancer.

HSP110 is induced by different stresses and, through its anti-apoptotic and chaperoning properties, helps the cells to survive these adverse situations. In colon cancers, HSP110 is abnormally abundant. We have recently showed that colorectal cancer (CRC) patients with microsatellite instability (MSI) had an improved response to chemotherapy because they harbor an HSP110 inactivating mutation (HSP110DE9). In this work, we have used patients' biopsies and human CRC cells grown in vitro and in vivo (xenografts) to demonstrate that (1) HSP110 is secreted by CRC cells and that the amount of this extracellular HSP110 is strongly decreased by the expression of the mutant HSP110DE9, (2) Supernatants from CRC cells overexpressing HSP110 or purified recombinant human HSP110 (LPS-free) affect macrophage differentiation/polarization by favoring a pro-tumor, anti-inflammatory profile, (3) Conversely, inhibition of HSP110 (expression of siRNA, HSP110DE9 or immunodepletion) induced the formation of macrophages with a cytotoxic, pro-inflammatory profile. (4) Finally, this effect of extracellular HSP110 on macrophages seems to implicate TLR4. These results together with the fact that colorectal tumor biopsies with HSP110 high were infiltrated with macrophages with a pro-tumoral profile while those with HSP110 low were infiltrated with macrophages with a cytotoxic profile, suggest that the effect of extracellular HSP110 function on macrophages may also contribute to the poor outcomes associated with HSP110 expression.

Heat shock proteins in fibrosis and wound healing: good or evil?

Heat shock proteins (HSPs) are key regulators of cell homeostasis, and their cytoprotective role has been largely investigated in the last few decades. However, an increasing amount of evidence highlights their deleterious effects on several human pathologies, including cancer, in which they promote tumor cell survival, proliferation and drug resistance. Therefore, HSPs have recently been suggested as therapeutic targets for improving human disease outcomes. Fibrotic diseases and cancer share several properties; both pathologies are characterized by genetic alterations, uncontrolled cell proliferation, altered cell interactions and communication and tissue invasion. The discovery of new HSP inhibitors that have been shown to be efficacious against certain types of cancers has given rise to a new field of research that investigates the activity of these compounds in other incurable human diseases such as fibrotic disorders. The aim of this review is to discuss new findings regarding the involvement of HSPs in the pathogenesis of organ fibrosis and to note recent discoveries that indicate that HSPs could be important therapeutic targets to improve the current dismal outcome of fibrotic diseases.

The small heat-shock protein αB-crystallin is essential for the nuclear localization of Smad4: impact on pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a devastating disease characterized by the proliferation of myofibroblasts and the accumulation of extracellular matrix (ECM) in the lungs. TGF-ß1 is the major profibrotic cytokine involved in IPF and is responsible for myofibroblast proliferation and differentiation and ECM synthesis. αB-crystallin is constitutively expressed in the lungs and is inducible by stress, acts as a chaperone and is known to play a role in cell cytoskeleton architecture homeostasis. The role of αB-crystallin in fibrogenesis remains unknown. The principal signalling pathway involved in this process is the Smad-dependent pathway. We demonstrate here that αB-crystallin is strongly expressed in fibrotic lung tissue from IPF patients and in vivo rodent models of pulmonary fibrosis. We also show that αB-crystallin-deficient mice are protected from bleomycin-induced fibrosis. Similar protection from fibrosis was observed in αB-crystallin KO mice after transient adenoviral-mediated over-expression of IL-1ß or TGF-ß1. We show in vitro in primary epithelial cells and fibroblasts that αB-crystallin increases the nuclear localization of Smad4, thereby enhancing the TGF-ß1-Smad pathway and the consequent activation of TGF-ß1 downstream genes. αB-crystallin over-expression disrupts Smad4 mono-ubiquitination by interacting with its E3-ubiquitin ligase, TIF1γ, thus limiting its nuclear export. Conversely, in the absence of αB-crystallin, TIF1γ can freely interact with Smad4. Consequently, Smad4 mono-ubiquitination and nuclear export are favoured and thus TGF-ß1-Smad4 pro-fibrotic activity is inhibited. This study demonstrates that αB-crystallin may be a key target for the development of specific drugs in the treatment of IPF or other fibrotic diseases.

High-frequency promoter firing links THO complex function to heavy chromatin formation.

The THO complex is involved in transcription, genome stability, and messenger ribonucleoprotein (mRNP) formation, but its precise molecular function remains enigmatic. Under heat shock conditions, THO mutants accumulate large protein-DNA complexes that alter the chromatin density of target genes (heavy chromatin), defining a specific biochemical facet of THO function and a powerful tool of analysis. Here, we show that heavy chromatin distribution is dictated by gene boundaries and that the gene promoter is necessary and sufficient to convey THO sensitivity in these conditions. Single-molecule fluorescence in situ hybridization measurements show that heavy chromatin formation correlates with an unusually high firing pace of the promoter with more than 20 transcription events per minute. Heavy chromatin formation closely follows the modulation of promoter firing and strongly correlates with polymerase occupancy genome wide. We propose that the THO complex is required for tuning the dynamic of gene-nuclear pore association and mRNP release to the same high pace of transcription initiation.

Real-time dynamics of RNA polymerase II clustering in live human cells.

Transcription is reported to be spatially compartmentalized in nuclear transcription factories with clusters of RNA polymerase II (Pol II). However, little is known about when these foci assemble or their relative stability. We developed a quantitative single-cell approach to characterize protein spatiotemporal organization, with single-molecule sensitivity in live eukaryotic cells. We observed that Pol II clusters form transiently, with an average lifetime of 5.1 (± 0.4) seconds, which refutes the notion that they are statically assembled substructures. Stimuli affecting transcription yielded orders-of-magnitude changes in the dynamics of Pol II clusters, which implies that clustering is regulated and plays a role in the cell's ability to effect rapid response to external signals. Our results suggest that transient crowding of enzymes may aid in rate-limiting steps of gene regulation.

Gene expression is circular: factors for mRNA degradation also foster mRNA synthesis.

Maintaining proper mRNA levels is a key aspect in the regulation of gene expression. The balance between mRNA synthesis and decay determines these levels. We demonstrate that most yeast mRNAs are degraded by the cytoplasmic 5'-to-3' pathway (the "decaysome"), as proposed previously. Unexpectedly, the level of these mRNAs is highly robust to perturbations in this major pathway because defects in various decaysome components lead to transcription downregulation. Moreover, these components shuttle between the cytoplasm and the nucleus, in a manner dependent on proper mRNA degradation. In the nucleus, they associate with chromatin-preferentially ∼30 bp upstream of transcription start-sites-and directly stimulate transcription initiation and elongation. The nuclear role of the decaysome in transcription is linked to its cytoplasmic role in mRNA decay; linkage, in turn, seems to depend on proper shuttling of its components. The gene expression process is therefore circular, whereby the hitherto first and last stages are interconnected.

The in vivo kinetics of RNA polymerase II elongation during co-transcriptional splicing.

RNA processing events that take place on the transcribed pre-mRNA include capping, splicing, editing, 3' processing, and polyadenylation. Most of these processes occur co-transcriptionally while the RNA polymerase II (Pol II) enzyme is engaged in transcriptional elongation. How Pol II elongation rates are influenced by splicing is not well understood. We generated a family of inducible gene constructs containing increasing numbers of introns and exons, which were stably integrated in human cells to serve as actively transcribing gene loci. By monitoring the association of the transcription and splicing machineries on these genes in vivo, we showed that only U1 snRNP localized to the intronless gene, consistent with a splicing-independent role for U1 snRNP in transcription. In contrast, all snRNPs accumulated on intron-containing genes, and increasing the number of introns increased the amount of spliceosome components recruited. This indicates that nascent RNA can assemble multiple spliceosomes simultaneously. Kinetic measurements of Pol II elongation in vivo, Pol II ChIP, as well as use of Spliceostatin and Meayamycin splicing inhibitors showed that polymerase elongation rates were uncoupled from ongoing splicing. This study shows that transcription elongation kinetics proceed independently of splicing at the model genes studied here. Surprisingly, retention of polyadenylated mRNA was detected at the transcription site after transcription termination. This suggests that the polymerase is released from chromatin prior to the completion of splicing, and the pre-mRNA is post-transcriptionally processed while still tethered to chromatin near the gene end.