RNA Polymerase II-associated Protein Research Articles

Identification and characterization of both cis- and trans-regulators mediating fenvalerate-induced expression of CYP6B7 in Helicoverpa armigera

As we known, inducibility is an important feature of P450 genes. Previous studies indicated that CYP6B7 could be induced and involved in fenvalerate detoxification in Helicoverpa armigera. However, the regulatory mechanism of CYP6B7 induced by fenvalerate is still unclear. In this study, CYP6B7 promoter of H. armigera was cloned and the cis-acting element of fenvalerate was identified to be located between −84 and − 55 bp of CYP6B7 promoter. Subsequently, 33 candidate transcription factors (CYP6B7-fenvalerate association proteins, CAPs) that may bind to the cis-acting element were screened and verified by yeast one-hybrid. Among them, the expression levels of several CAPs were significantly induced by fenvalerate. Knockdown of juvenile hormone-binding protein-like (JHBP), RNA polymerase II-associated protein 3 (RPAP3), fatty acid synthase-like (FAS) and peptidoglycan recognition protein LB-like (PGRP) resulted in significant expression inhibition of CYP6B7, and increased sensitivity of H. armigera to fenvalerate. Co-transfection of reporter gene p (−84/−55) with pFast-CAP showed that JHBP, RPAP3 and PGRP could significantly increase the activity of CYP6B7 promoter. These results suggested that trans-acting factors JHBP, RPAP3 and PGRP may bind with cis-acting elements to regulate the expression of CYP6B7 induced by fenvalerate, and play an important role in the detoxification of H. armigera to fenvalerate.

Super enhancers targeting ZBTB16 in osteogenesis protect against osteoporosis

As the major cell precursors in osteogenesis, mesenchymal stem cells (MSCs) are indispensable for bone homeostasis and development. However, the primary mechanisms regulating osteogenic differentiation are controversial. Composed of multiple constituent enhancers, super enhancers (SEs) are powerful cis-regulatory elements that identify genes that ensure sequential differentiation. The present study demonstrated that SEs were indispensable for MSC osteogenesis and involved in osteoporosis development. Through integrated analysis, we identified the most common SE-targeted and osteoporosis-related osteogenic gene, ZBTB16. ZBTB16, positively regulated by SEs, promoted MSC osteogenesis but was expressed at lower levels in osteoporosis. Mechanistically, SEs recruited bromodomain containing 4 (BRD4) at the site of ZBTB16, which then bound to RNA polymerase II-associated protein 2 (RPAP2) that transported RNA polymerase II (POL II) into the nucleus. The subsequent synergistic regulation of POL II carboxyterminal domain (CTD) phosphorylation by BRD4 and RPAP2 initiated ZBTB16 transcriptional elongation, which facilitated MSC osteogenesis via the key osteogenic transcription factor SP7. Bone-targeting ZBTB16 overexpression had a therapeutic effect on the decreased bone density and remodeling capacity of Brd4fl/flPrx1-cre mice and osteoporosis (OP) models. Therefore, our study shows that SEs orchestrate the osteogenesis of MSCs by targeting ZBTB16 expression, which provides an attractive focus and therapeutic target for osteoporosis.Without SEs located on osteogenic genes, BRD4 is not able to bind to osteogenic identity genes due to its closed structure before osteogenesis. During osteogenesis, histones on osteogenic identity genes are acetylated, and OB-gain SEs appear, enabling the binding of BRD4 to the osteogenic identity gene ZBTB16. RPAP2 transports RNA Pol II from the cytoplasm to the nucleus and guides Pol II to target ZBTB16 via recognition of the navigator BRD4 on SEs. After the binding of the RPAP2-Pol II complex to BRD4 on SEs, RPAP2 dephosphorylates Ser5 at the Pol II CTD to terminate the transcriptional pause, and BRD4 phosphorylates Ser2 at the Pol II CTD to initiate transcriptional elongation, which synergistically drives efficient transcription of ZBTB16, ensuring proper osteogenesis. Dysregulation of SE-mediated ZBTB16 expression leads to osteoporosis, and bone-targeting ZBTB16 overexpression is efficient in accelerating bone repair and treating osteoporosis.

RNA polymerase II-associated proteins reveal pathways affected in VCP-related amyotrophic lateral sclerosis.

Valosin-containing protein (VCP) is a hexameric ATPase associated with diverse cellular activities. Genetic mutations in VCP are associated with several forms of muscular and neuronal degeneration, including amyotrophic lateral sclerosis (ALS). Moreover, VCP mediates UV-induced proteolysis of RNA polymerase II (RNAPII), but little is known about the effects of VCP mutations on the transcriptional machinery. Here, we used silica particle-assisted chromatin enrichment and mass spectrometry to study proteins co-localized with RNAPII in precursor neurons differentiated from VCP-mutant or control induced pluripotent stem cells. Remarkably, we observed diminished RNAPII binding of proteins involved in transcription elongation and mRNA splicing in mutant cells. One of these is SART3, a recycling factor of the splicing machinery, whose knockdown leads to perturbed intron retention in several ALS-associated genes. Additional reduced proteins are RBM45, EIF5A and RNF220, mutations in which are associated with various neurodegenerative disorders and are linked to TDP-43 aggregation. Conversely, we observed increased RNAPII binding of heat shock proteins such as HSPB1. Together, these findings shed light on how transcription and splicing machinery are impaired by VCP mutations, which might contribute to aberrant alternative splicing and proteinopathy in neurodegeneration.

RPAP3 C-Terminal Domain: A Conserved Domain for the Assembly of R2TP Co-Chaperone Complexes.

The Rvb1-Rvb2-Tah1-Pih1 (R2TP) complex is a co-chaperone complex that works together with HSP90 in the activation and assembly of several macromolecular complexes, including RNA polymerase II (Pol II) and complexes of the phosphatidylinositol-3-kinase-like family of kinases (PIKKs), such as mTORC1 and ATR/ATRIP. R2TP is made of four subunits: RuvB-like protein 1 (RUVBL1) and RuvB-like 2 (RUVBL2) AAA-type ATPases, RNA polymerase II-associated protein 3 (RPAP3), and the Protein interacting with Hsp90 1 (PIH1) domain-containing protein 1 (PIH1D1). R2TP associates with other proteins as part of a complex co-chaperone machinery involved in the assembly and maturation of a growing list of macromolecular complexes. Recent progress in the structural characterization of R2TP has revealed an alpha-helical domain at the C-terminus of RPAP3 that is essential to bring the RUVBL1 and RUVBL2 ATPases to R2TP. The RPAP3 C-terminal domain interacts directly with RUVBL2 and it is also known as RUVBL2-binding domain (RBD). Several human proteins contain a region homologous to the RPAP3 C-terminal domain, and some are capable of assembling R2TP-like complexes, which could have specialized functions. Only the RUVBL1-RUVBL2 ATPase complex and a protein containing an RPAP3 C-terminal-like domain are found in all R2TP and R2TP-like complexes. Therefore, the RPAP3 C-terminal domain is one of few components essential for the formation of all R2TP and R2TP-like co-chaperone complexes.

Target Identification of the Marine Natural Products Dictyoceratin-A and -C as Selective Growth Inhibitors in Cancer Cells Adapted to Hypoxic Environments

Hypoxia-adapted cancer cells in tumors contribute to the pathological progression of cancer. The marine spongean sesquiterpene phenols dictyoceratin-A (1) and -C (2) have been shown to induce hypoxia-selective growth inhibition in cultured cancer cells and exhibit in vivo antitumor effects. These compounds inhibit the accumulation of hypoxia-inducible factor-1α (HIF-1α), which is a drug target in hypoxia-adapted cancer cells, under hypoxic conditions. However, the target molecules of compounds 1 and 2, which are responsible for decreasing HIF-1α expression under hypoxic conditions, remain unclear. In this study, we synthesized probe molecules for compounds 1 and 2 to identify their target molecules and found that both compounds bind to RNA polymerase II-associated protein 3 (RPAP3), which is a component of the R2TP/Prefoldin-like (PEDL) complex. In addition, RPAP3-knockdown cells showed a phenotype similar to that of compound-treated cells.

Coordination between Two Branches of the Unfolded Protein Response Determines Apoptotic Cell Fate

The kinases PERK and IRE1 alleviate endoplasmic reticulum (ER) stress by orchestrating the unfolded protein response (UPR). If stress mitigation fails, PERK promotes cell death by activating pro-apoptotic genes, including death receptor 5 (DR5). Conversely, IRE1-which harbors both kinase and endoribonuclease (RNase) modules-blocks apoptosis through regulated IRE1-dependent decay (RIDD) of DR5 mRNA. Under irresolvable ER stress, PERK activity persists, whereas IRE1 paradoxically attenuates, by mechanisms that remain obscure. Here, we report that PERK governs IRE1's attenuation through a phosphatase known as RPAP2 (RNA polymerase II-associated protein 2). RPAP2 reverses IRE1 phosphorylation, oligomerization, and RNase activation. This inhibits IRE1-mediated adaptive events, including activation of the cytoprotective transcription factor XBP1s, and ER-associated degradation of unfolded proteins. Furthermore, RIDD termination by RPAP2 unleashes DR5-mediated caspase activation and drives cell death. Thus, PERK attenuates IRE1 via RPAP2 to abort failed ER-stress adaptation and trigger apoptosis.

The RNA Polymerase II Factor RPAP1 Is Critical for Mediator-Driven Transcription and Cell Identity

SummaryThe RNA polymerase II-associated protein 1 (RPAP1) is conserved across metazoa and required for stem cell differentiation in plants; however, very little is known about its mechanism of action or its role in mammalian cells. Here, we report that RPAP1 is essential for the expression of cell identity genes and for cell viability. Depletion of RPAP1 triggers cell de-differentiation, facilitates reprogramming toward pluripotency, and impairs differentiation. Mechanistically, we show that RPAP1 is essential for the interaction between RNA polymerase II (RNA Pol II) and Mediator, as well as for the recruitment of important regulators, such as the Mediator-specific RNA Pol II factor Gdown1 and the C-terminal domain (CTD) phosphatase RPAP2. In agreement, depletion of RPAP1 diminishes the loading of total and Ser5-phosphorylated RNA Pol II on many genes, with super-enhancer-driven genes among the most significantly downregulated. We conclude that Mediator/RPAP1/RNA Pol II is an ancient module, conserved from plants to mammals, critical for establishing and maintaining cell identity.

Spaghetti, a homolog of human RPAP3 (RNA polymerase II-associated protein 3), determines the fate of germline stem cells in Drosophila ovary.

The Drosophila ovary provides an attractive model for studying the extrinsic or intrinsic factors that regulate the fate of germline stem cells (GSCs). Using this model, we identified a new role for Drosophila spaghetti (spag), encoding a homolog of human RNA polymerase II-associated protein 3 (RPAP3), in regulating the fate of ovarian GSCs. Results from spag knockdown and genetic mosaic studies suggest that spag functions as an intrinsic factor for GSCs maintenance. Loss of Spag by, either spag RNAi or null mutation failed to trigger apoptosis in ovarian GSCs. Overexpression of spag led to negligible increases in the number of GSC/Cystoblast (CB) cells, suggesting that an excess of Spag is not sufficient to accelerate the proliferation of GSCs or delay CBs' differentiation. Our study provides evidence supporting that spag is involved in adult stem cells maintenance. In addition, the RNAi screen results showed that knockdown of Hsp90, one of known Spag interacting partners, led to loss of ovarian GSCs in Drosophila. Heterozygous mutations in hsp90 (hsp90/+) dramatically accelerated the GSC loss in spag RNAi ovaries, suggesting that the Spag-contained complex possibly plays an essential role in controlling the GSCs fate.

A genome-wide IR-induced RAD51 foci RNAi screen identifies CDC73 involved in chromatin remodeling for DNA repair.

To identify new regulators of homologous recombination repair, we carried out a genome-wide short-interfering RNA screen combined with ionizing irradiation using RAD51 foci formation as readout. All candidates were confirmed by independent short-interfering RNAs and validated in secondary assays like recombination repair activity and RPA foci formation. Network analysis of the top modifiers identified gene clusters involved in recombination repair as well as components of the ribosome, the proteasome and the spliceosome, which are known to be required for effective DNA repair. We identified and characterized the RNA polymerase II-associated protein CDC73/Parafibromin as a new player in recombination repair and show that it is critical for genomic stability. CDC73 interacts with components of the SCF/Cullin and INO80/NuA4 chromatin-remodeling complexes to promote Histone ubiquitination. Our findings indicate that CDC73 is involved in local chromatin decondensation at sites of DNA damage to promote DNA repair. This function of CDC73 is related to but independent of its role in transcriptional elongation.

Abnormal expression of RNA polymerase II-associated proteins in muscle of patients with myofibrillar myopathies.

Myofibrillar myopathies (MFMs) are a group of inherited or sporadic neuromuscular disorders characterized morphologically by foci of myofibril dissolution, disintegration of the Z-disk and insoluble protein aggregates within the muscle fibres. The sequential events leading to muscle fibre damage remains largely unknown. We investigated the expression and the cellular localization of RNA polymerase II (RNAPII)-associated proteins (RPAPs) in muscle biopsies from patients with genetically proven and sporadic MFMs. Our data demonstrated that RPAP2, and to a lesser extent GPN1/RPAP4, are accumulated focally in the cytoplasm of MFM muscle fibres in which they co-localize with POLR2A/RPB1, the largest subunit of RNAPII, and correspond to αB-cystallin deposits in distribution and staining intensity. No abnormal staining for RPAP2 has been observed in muscle of patients with central cores, minicores and neurogenic target fibres. Together, these findings could provide new insights into the molecular pathogenesis of MFMs and suggest that RPAP2 immunostaining can be a useful diagnostic tool to depict protein aggregates in MFMs.

Drosophila Spag Is the Homolog of RNA Polymerase II-associated Protein 3 (RPAP3) and Recruits the Heat Shock Proteins 70 and 90 (Hsp70 and Hsp90) during the Assembly of Cellular Machineries

The R2TP is a recently identified Hsp90 co-chaperone, composed of four proteins as follows: Pih1D1, RPAP3, and the AAA(+)-ATPases RUVBL1 and RUVBL2. In mammals, the R2TP is involved in the biogenesis of cellular machineries such as RNA polymerases, small nucleolar ribonucleoparticles and phosphatidylinositol 3-kinase-related kinases. Here, we characterize the spaghetti (spag) gene of Drosophila, the homolog of human RPAP3. This gene plays an essential function during Drosophila development. We show that Spag protein binds Drosophila orthologs of R2TP components and Hsp90, like its yeast counterpart. Unexpectedly, Spag also interacts and stimulates the chaperone activity of Hsp70. Using null mutants and flies with inducible RNAi, we show that spaghetti is necessary for the stabilization of snoRNP core proteins and target of rapamycin activity and likely the assembly of RNA polymerase II. This work highlights the strong conservation of both the HSP90/R2TP system and its clients and further shows that Spag, unlike Saccharomyces cerevisiae Tah1, performs essential functions in metazoans. Interaction of Spag with both Hsp70 and Hsp90 suggests a model whereby R2TP would accompany clients from Hsp70 to Hsp90 to facilitate their assembly into macromolecular complexes.

Human RNA polymerase II-associated protein 2 (RPAP2) interacts directly with the RNA polymerase II subunit Rpb6 and participates in pre-mRNA 3'-end formation.

The C-terminal domain (CTD) of the largest subunit of RNA polymerase II (Pol II) is composed of tandem repeats of the heptapeptide Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7. The CTD of Pol II undergoes reversible phosphorylation during the transcription cycle, mainly at Ser2, Ser5, and Ser7. Dynamic changes in the phosphorylation patterns of the CTD are responsible for stage-specific recruitment of various factors involved in RNA processing, histone modification, and transcription elongation/termination. Human RNA polymerase II-associated protein 2 (RPAP2) was originally identified as a Pol II-associated protein and was subsequently shown to function as a novel Ser5-specific CTD phosphatase. Although a recent study suggested that RPAP2 is required for the efficient expression of small nuclear RNA genes, the role of RPAP2 in controlling the expression of protein-coding genes is unknown. Here, we demonstrate that the C-terminal region of RPAP2 interacts directly with the Pol II subunit Rpb6. Chromatin immunoprecipitation analyses of the MYC and GAPDH protein-coding genes revealed that RPAP2 occupied the coding and 3' regions. Notably, siRNA-mediated knockdown of RPAP2 caused defects in 3'-end formation of the MYC and GAPDH pre-mRNAs. These results suggest that RPAP2 controls Pol II activity through a direct interaction with Rpb6 and participates in pre-mRNA 3'-end formation.

Nuclear import of RNA polymerase II is coupled with nucleocytoplasmic shuttling of the RNA polymerase II-associated protein 2

The RNA polymerase II (RNAP II)-associated protein (RPAP) 2 has been discovered through its association with various subunits of RNAP II in affinity purification coupled with mass spectrometry experiments. Here, we show that RPAP2 is a mainly cytoplasmic protein that shuttles between the cytoplasm and the nucleus. RPAP2 shuttling is tightly coupled with nuclear import of RNAP II, as RPAP2 silencing provokes abnormal accumulation of RNAP II in the cytoplasmic space. Most notably, RPAP4/GPN1 silencing provokes the retention of RPAP2 in the nucleus. Our results support a model in which RPAP2 enters the nucleus in association with RNAP II and returns to the cytoplasm in association with the GTPase GPN1/RPAP4. Although binding of RNAP II to RPAP2 is mediated by an N-terminal domain (amino acids 1–170) that contains a nuclear retention domain, and binding of RPAP4/GPN1 to RPAP2 occurs through a C-terminal domain (amino acids 156–612) that has a dominant cytoplasmic localization domain. In conjunction with previously published data, our results have important implications, as they indicate that RPAP2 controls gene expression by two distinct mechanisms, one that targets RNAP II activity during transcription and the other that controls availability of RNAP II in the nucleus.

RPAP3 splicing variant isoform 1 interacts with PIH1D1 to compose R2TP complex for cell survival

We previously characterized RNA polymerase II-associated protein 3 (RPAP3) as a cell death enhancer. Here we report the identification and characterization of splicing isoform of RPAP3, isoform 1 and 2. We investigated the interaction between RPAP3 and PIH1 domain containing protein 1 (PIH1D1), and found that RPAP3 isoform 1, but not isoform 2, interacted with PIH1D1. Furthermore, knockdown of RPAP3 isoform 1 by small interfering RNA down-regulated PIH1D1 protein level without affecting PIH1D1 mRNA. RPAP3 isoform 2 potentiated doxorubicin-induced cell death in human breast cancer T-47 cells although isoform 1 showed no effect. These results suggest that R2TP complex is composed of RPAP3 isoform 1 for its stabilization, and that RPAP3 isoform 2 may have a dominant negative effect on the survival potency of R2TP complex.

A two-stage association study identifies methyl-CpG-binding domain protein 2 gene polymorphisms as candidates for breast cancer susceptibility

Genome-wide association studies for breast cancer have identified over 40 single-nucleotide polymorphisms (SNPs), a subset of which remains statistically significant after genome-wide correction. Improved strategies for mining of genome-wide association data have been suggested to address heritable component of genetic risk in breast cancer. In this study, we attempted a two-stage association design using markers from a genome-wide study (stage 1, Affymetrix Human SNP 6.0 array, cases=302, controls=321). We restricted our analysis to DNA repair/modifications/metabolism pathway related gene polymorphisms for their obvious role in carcinogenesis in general and for their known protein–protein interactions vis-à-vis, potential epistatic effects. We selected 22 SNPs based on linkage disequilibrium patterns and high statistical significance. Genotyping assays in an independent replication study of 1178 cases and 1314 controls were attempted using Sequenom iPLEX Gold platform (stage 2). Six SNPs (rs8094493, rs4041245, rs7614, rs13250873, rs1556459 and rs2297381) showed consistent and statistically significant associations with breast cancer risk in both stages, with allelic odds ratios (and P-values) of 0.85 (0.0021), 0.86 (0.0026), 0.86 (0.0041), 1.17 (0.0043), 1.20 (0.0103) and 1.13 (0.0154), respectively, in combined analysis (N=3115). Of these, three polymorphisms were located in methyl-CpG-binding domain protein 2 gene regions and were in strong linkage disequilibrium. The remaining three SNPs were in proximity to RAD21 homolog (S. pombe), O-6-methylguanine-DNA methyltransferase and RNA polymerase II-associated protein 1. The identified markers may be relevant to breast cancer susceptibility in populations if these findings are confirmed in independent cohorts.

RPAP3 enhances cytotoxicity of doxorubicin by impairing NF-kappa B pathway

Activation of anti-apoptotic gene transcription by NF-κB (nuclear factor-kappa B) has been reported to be linked with a resistance of cancer cells against chemotherapy. NEMO (NF-κB essential modulator) interacts with a number of proteins and modulates the activity of NF-κB pathway. In this study, we revealed that RPAP3 (RNA polymerase II-associated protein 3) possesses an activity to bind with NEMO and to inhibit the ubiquitination of NEMO and that RPAP3 enhances doxorubicin-induced cell death in breast cancer cell line T-47D through the marked impairment of NF-κB pathway. These results indicate that RPAP3 may be a novel modulator of NF-κB pathway in apoptosis induced by anti-cancer chemotherapeutic agents.

PIH1D1, a subunit of R2TP complex, inhibits doxorubicin-induced apoptosis

We have previously reported that the two components of R2TP complex, RNA polymerase II-associated protein 3 (RPAP3), and Reptin, regulate apoptosis. Here we characterize another component of the complex, PIH1 domain containing protein 1 (PIH1D1). PIH1D1 interacts with both RPAP3 and Monad in HEK293 or U2OS cells. PIH1D1 transcripts were abundant in lung, leukocyte, and placenta. The reduction in endogenous PIH1D1 by siRNA enhanced apoptosis and caspase-3 activation induced by doxorubicin in U2OS cells. These results suggest that PIH1D1 may also function as a novel modulator of apoptosis pathway.

RPAP3 interacts with Reptin to regulate UV‐induced phosphorylation of H2AX and DNA damage

We have previously reported that Monad, a novel WD40 repeat protein, potentiates apoptosis induced by tumor necrosis factor-alpha and cycloheximide. By affinity purification and mass spectrometry, RNA polymerase II-associated protein 3 (RPAP3) was identified as a Monad binding protein and may function with Monad as a novel modulator of apoptosis pathways. Here we report that Reptin, a highly conserved AAA + ATPase that is part of various chromatin-remodeling complexes, is also involved in the association of RPAP3 by immunoprecipitation and confocal microscopic analysis. Overexpression of RPAP3 induced HEK293 cells to death after UV-irradiation. Loss of RPAP3 by RNAi improved HeLa cell survival after UV-induced DNA damage and attenuated the phosphorylation of H2AX. Depletion of Reptin reduced cell survival and facilitated the phosphorylation on H2AX. These results suggest that RPAP3 modulates UV-induced DNA damage by regulating H2AX phosphorylation.

Molecular cloning of novel Monad binding protein containing tetratricopeptide repeat domains

We have previously reported that Monad, a novel WD40 repeat protein, potentiates apoptosis induced by tumor necrosis factor-α(TNF-α) and cycloheximide (CHX). By affinity purification and mass spectrometry, we identified RNA polymerase II-associated protein 3 (RPAP3) as a binding protein of Monad. Overexpression of RPAP3 in HEK 293 potentiated caspase-3 activation and apoptosis induced by TNF-α and CHX. In addition, knockdown of RPAP3 by RNA interference resulted in a significant reduction of apoptosis induced by TNF-α and CHX in HEK293 and HeLa cells. These results raise the possibility that RPAP3, together with Monad, may function as a novel modulator of apoptosis pathway. Structured summary MINT- 6551090: Monad (uniprotkb: Q96MX6) physically interacts (MI: 0218) with RPAP3 (uniprotkb: Q9H6T3) by anti tag coimmunoprecipitation (MI: 0007) MINT- 6551101, MINT- 6551118: Monad (uniprotkb: Q96MX6) physically interacts (MI: 0218) with RPAP3 (uniprotkb: Q9H6T3) by pull down (MI: 0096) MINT- 6551132: RPAP3 (uniprotkb: Q9H6T3) physically interacts (MI: 0218) with Monad (uniprotkb: Q96MX6) by anti bait coimmunoprecipitation (MI: 0006)

Cotranscriptional Recruitment of the Pseudouridylsynthetase Cbf5p and of the RNA Binding Protein Naf1p during H/ACA snoRNP Assembly

H/ACA small nucleolar ribonucleoprotein particles (snoRNPs) are essential for the maturation and pseudouridylation of the precursor of rRNAs and other stable RNAs. Although the RNA and protein components of these RNPs have been identified, the mechanisms by which they are assembled in vivo are poorly understood. Here we show that the RNA binding protein Naf1p, which is required for H/ACA snoRNPs stability, associates with RNA polymerase II-associated proteins Spt16p, Tfg1p, and Sub1p and with H/ACA snoRNP proteins. Chromatin immunoprecipitation experiments show that Naf1p and the pseudouridylsynthetase Cbf5p cross-link specifically with the chromatin of H/ACA small nucleolar RNA (snoRNA) genes. Naf1p and Cbf5p cross-link predominantly with the 3' end of these genes, in a pattern similar to that observed for transcription elongation factor Spt16p. Cross-linking of Naf1p to H/ACA snoRNA genes requires active transcription and intact H/ACA snoRNA sequences but does not require the RNA polymerase II CTD kinase Ctk1p. These results suggest that Naf1p and Cbf5p are recruited in a cotranscriptional manner during H/ACA snoRNP assembly, possibly by binding to the nascent H/ACA snoRNA transcript during elongation or termination of transcription of H/ACA snoRNA genes.