Osteopontin and thrombospondin-1 play opposite roles in promoting tumor aggressiveness of primary resected non-small cell lung cancer.

BACKGROUND: Osteopontin (OPN) and thrombospondin-1 (TSP-1) are extracellular matrix proteins secreted by stromal and tumor cells. These proteins appear to have a key role in the tumor microenvironment for cancer development and metastasis. There is little information regarding the prognostic value of the combination of these two proteins in human cancers. Our aim was to clarify clinical significance and prognostic value of each circulating protein and their combination in primary resected non-small cell lung cancer (NSCLC) patients. METHODS: We retrospectively reviewed 171 patients with NSCLC following curative intent surgery from January to December of 2012. Preoperative serums, demographics, clinical and pathological data and molecular profiling were analyzed. Pre-treatment OPN and TSP-1 serum levels were measured by ELISA. Tissue protein expression in primary tumor samples was determined by immunohistochemical analysis. RESULTS: OPN and TSP-1 serum levels were inversely correlated with survival rates. For each 50 units increment of serum OPN, an increased risk of metastasis by 69 % (unadjusted HR 1.69, 95 % CI 1.12-2.56, p = 0.01) and an increased risk of death by 95 % (unadjusted HR 1.95, 95 % CI 1.15-3.32, p = 0.01) were observed. Conversely, for each 10 units increment in TSP-1, the risk of death was decreased by 85 % (unadjusted HR 0.15, 95 % CI 0.03-0.89; p = 0.04). No statistically significant correlation was found between TSP-1 serum level and distant metastasis-free survival (p = 0.2). On multivariate analysis, OPN and TSP-1 serum levels were independent prognostic factors of overall survival (HR 1.71, 95 % CI 1.04-2.82, p = 0.04 for an increase of 50 ng/mL in OPN; HR 0.18, 95 % CI 0.04-0.87, p = 0.03 for an increase of 10 ng/mL in TSP-1). In addition, the combination of OPN and TSP-1 serum levels remained an independent prognostic factor for overall survival (HR 1.31, 95 % CI 1.03-1.67, p = 0.03 for an increase of 6 ng/mL in OPN/TSP-1 ratio). CONCLUSIONS: Our results show that pre-treatment OPN and TSP-1 serum levels may reflect the aggressiveness of the tumor and might serve as prognostic markers in patients with primary resected NSCLC.

Nuclear myosin 1c facilitates the chromatin modifications required to activate rRNA gene transcription and cell cycle progression.

Actin and nuclear myosin 1c (NM1) cooperate in RNA polymerase I (pol I) transcription. NM1 is also part of a multiprotein assembly, B-WICH, which is involved in transcription. This assembly contains the chromatin remodeling complex WICH with its subunits WSTF and SNF2h. We report here that NM1 binds SNF2h with enhanced affinity upon impairment of the actin-binding function. ChIP analysis revealed that NM1, SNF2h, and actin gene occupancies are cell cycle-dependent and require intact motor function. At the onset of cell division, when transcription is temporarily blocked, B-WICH is disassembled due to WSTF phosphorylation, to be reassembled on the active gene at exit from mitosis. NM1 gene knockdown and motor function inhibition, or stable expression of NM1 mutants that do not interact with actin or chromatin, overall repressed rRNA synthesis by stalling pol I at the gene promoter, led to chromatin alterations by changing the state of H3K9 acetylation at gene promoter, and delayed cell cycle progression. These results suggest a unique structural role for NM1 in which the interaction with SNF2h stabilizes B-WICH at the gene promoter and facilitates recruitment of the HAT PCAF. This leads to a permissive chromatin structure required for transcription activation.

Nucleolar scaffold protein, WDR46, determines the granular compartmental localization of nucleolin and DDX21.

The nuclear scaffold is an insoluble nuclear structure that contributes to the inner nuclear organization. In this study, we showed that one of the nuclear scaffold proteins, WDR46, plays a role as a fundamental scaffold component of the nucleolar structure. WDR46 is a highly insoluble nucleolar protein, and its subcellular localization is dependent on neither DNA nor RNA. The N- and C-terminal regions of WDR46 are predicted to be intrinsically disordered, and both regions are critical for the nucleolar localization of WDR46 and the association with its binding partners. When WDR46 was knocked down, two of its binding partners, nucleolin and DDX21 (involved in 18S rRNA processing), were mislocalized from the granular component to the edges of the nucleoli, whereas other binding partners, NOP2 and EBP2 (involved in 28S rRNA processing), were not affected. This is because the proper recruitment of nucleolin and DDX21 to the nucleoli in daughter cells after cell division is ensured by WDR46. These findings suggest a structural role for WDR46 in organizing the 18S ribosomal RNA processing machinery. This role of WDR46 is enabled by its interaction property via intrinsically disordered regions.

Probing the stiffness of isolated nucleoli by atomic force microscopy.

In eukaryotic cells, ribosome biogenesis occurs in the nucleolus, a membraneless nuclear compartment. Noticeably, the nucleolus is also involved in several nuclear functions, such as cell cycle regulation, non-ribosomal ribonucleoprotein complex assembly, aggresome formation and some virus assembly. The most intriguing question about the nucleolus is how such dynamics processes can occur in such a compact compartment. We hypothesized that its structure may be rather flexible. To investigate this, we used atomic force microscopy (AFM) on isolated nucleoli. Surface topography imaging revealed the beaded structure of the nucleolar surface. With the AFM's ability to measure forces, we were able to determine the stiffness of isolated nucleoli. We could establish that the nucleolar stiffness varies upon drastic morphological changes induced by transcription and proteasome inhibition. Furthermore, upon ribosomal proteins and LaminB1 knockdowns, the nucleolar stiffness was increased. This led us to propose a model where the nucleolus has steady-state stiffness dependent on ribosome biogenesis activity and requires LaminB1 for its flexibility.

Identifying Patient Populations in Texts Describing Drug Approvals Through Deep Learning-Based Information Extraction: Development of a Natural Language Processing Algorithm.

BACKGROUND: New drug treatments are regularly approved, and it is challenging to remain up-to-date in this rapidly changing environment. Fast and accurate visualization is important to allow a global understanding of the drug market. Automation of this information extraction provides a helpful starting point for the subject matter expert, helps to mitigate human errors, and saves time. OBJECTIVE: We aimed to semiautomate disease population extraction from the free text of oncology drug approval descriptions from the BioMedTracker database for 6 selected drug targets. More specifically, we intended to extract (1) line of therapy, (2) stage of cancer of the patient population described in the approval, and (3) the clinical trials that provide evidence for the approval. We aimed to use these results in downstream applications, aiding the searchability of relevant content against related drug project sources. METHODS: We fine-tuned a state-of-the-art deep learning model, Bidirectional Encoder Representations from Transformers, for each of the 3 desired outputs. We independently applied rule-based text mining approaches. We compared the performances of deep learning and rule-based approaches and selected the best method, which was then applied to new entries. The results were manually curated by a subject matter expert and then used to train new models. RESULTS: The training data set is currently small (433 entries) and will enlarge over time when new approval descriptions become available or if a choice is made to take another drug target into account. The deep learning models achieved 61% and 56% 5-fold cross-validated accuracies for line of therapy and stage of cancer, respectively, which were treated as classification tasks. Trial identification is treated as a named entity recognition task, and the 5-fold cross-validated F1-score is currently 87%. Although the scores of the classification tasks could seem low, the models comprise 5 classes each, and such scores are a marked improvement when compared to random classification. Moreover, we expect improved performance as the input data set grows, since deep learning models need to be trained on a large enough amount of data to be able to learn the task they are taught. The rule-based approach achieved 60% and 74% 5-fold cross-validated accuracies for line of therapy and stage of cancer, respectively. No attempt was made to define a rule-based approach for trial identification. CONCLUSIONS: We developed a natural language processing algorithm that is currently assisting subject matter experts in disease population extraction, which supports health authority approvals. This algorithm achieves semiautomation, enabling subject matter experts to leverage the results for deeper analysis and to accelerate information retrieval in a crowded clinical environment such as oncology.

Nuclear myosin 1 is in complex with mature rRNA transcripts and associates with the nuclear pore basket.

In rRNA biogenesis, nuclear myosin 1 (NM1) and actin synergize to activate rRNA gene transcription. Evidence that actin is in preribosomal subunits and NM1 may control rRNA biogenesis post-transcriptionally prompted us to investigate whether NM1 associates with and accompanies rRNA to nuclear pores (NPC). Ultracentrifugation on HeLa nucleolar extracts showed RNA-dependent NM1 coelution with preribosomal subunits. In RNA immunoprecipitations (RIPs), NM1 coprecipitated with pre-rRNAs and 18S, 5.8S, and 28S rRNAs, but failed to precipitate 5S rRNA and 7SL RNA. In isolated nuclei and living HeLa cells, NM1 or actin inhibition and selective alterations in actin polymerization impaired 36S pre-rRNA processing. Immunoelectron microscopy (IEM) on sections of manually isolated Xenopus oocyte nuclei showed NM1 localization at the NPC basket. Field emission scanning IEM on isolated nuclear envelopes and intranuclear content confirmed basket localization and showed that NM1 decorates actin-rich pore-linked filaments. Finally, RIP and successive RIPs (reRIPs) on cross-linked HeLa cells demonstrated that NM1, CRM1, and Nup153 precipitate same 18S and 28S rRNAs but not 5S rRNA. We conclude that NM1 facilitates maturation and accompanies export-competent preribosomal subunits to the NPC, thus modulating export.

Adipose Tissue Properties in Tumor-Bearing Breasts.

The tissue stroma plays a major role in tumors' natural history. Most programs for tumor progression are not activated as cell-autonomous processes but under the conditions of cross-talks between tumor and stroma. Adipose tissue is a major component of breast stroma. This study compares adipose tissues in tumor-bearing breasts to those in tumor-free breasts with the intention of defining a signature that could translate into markers of cancer risk. In tumor-bearing breasts, we sampled adipose tissues adjacent to, or distant from the tumor. Parameters studied included: adipocytes size and density, immune cell infiltration, vascularization, secretome and gene expression. Adipose tissues from tumor-bearing breasts, whether adjacent to or distant from the tumor, do not differ from each other by any of these parameters. By contrast, adipose tissues from tumor-bearing breasts have the capacity to secrete twice as much interleukin 8 (IL-8) than those from tumor-free breasts and differentially express a set of 137 genes of which a significant fraction belongs to inflammation, integrin and wnt signaling pathways. These observations show that adipose tissues from tumor-bearing breasts have a distinct physiological status from those from tumor-free breasts. We propose that this constitutive status contributes as a non-cell autonomous process to determine permissiveness for tumor growth.

Compartmentation of the nucleolar processing proteins in the granular component is a CK2-driven process.

To analyze the compartmentation of nucleolar protein complexes, the mechanisms controlling targeting of nucleolar processing proteins onto rRNA transcription sites has been investigated. We studied the reversible disconnection of transcripts and processing proteins using digitonin-permeabilized cells in assays capable of promoting nucleolar reorganization. The assays show that the dynamics of nucleolar reformation is ATP/GTP-dependent, sensitive to temperature, and CK2-driven. We further demonstrate the role of CK2 on the rRNA-processing protein B23. Mutation of the major CK2 site on B23 induces reorganization of nucleolar components that separate from each other. This was confirmed in assays using extracts containing B23 mutated in the CK2-binding sites. We propose that phosphorylation controls the compartmentation of the rRNA-processing proteins and that CK2 is involved in this process.

Tracking the interactions of rRNA processing proteins during nucleolar assembly in living cells.

Reorganization of the nuclear machinery after mitosis is a fundamental but poorly understood process. Here, we investigate the recruitment of the nucleolar processing proteins in the nucleolus of living cells at the time of nucleus formation. We question the role of the prenucleolar bodies (PNBs), during migration of the processing proteins from the chromosome periphery to sites of rDNA transcription. Surprisingly, early and late processing proteins pass through the same PNBs as demonstrated by rapid two-color four-dimensional imaging and quantification, whereas a different order of processing protein recruitment into nucleoli is supported by differential sorting. Protein interactions along the recruitment pathway were investigated using a promising time-lapse analysis of fluorescence resonance energy transfer. For the first time, it was possible to detect in living cells the interactions between proteins of the same rRNA processing machinery in nucleoli. Interestingly interactions between such proteins also occur in PNBs but not at the chromosome periphery. The dynamics of these interactions suggests that PNBs are preassembly platforms for rRNA processing complexes.

TPF induction chemotherapy increases PD-L1 expression in tumour cells and immune cells in head and neck squamous cell carcinoma.

BACKGROUND: Antiprogrammed cell death-1/programmed cell death-ligand 1 (PD-1/PD-L1) therapies have demonstrated promising activity in advanced head and neck squamous cell carcinoma (HNSCC), with overall response rates of approximately 20% in unselected populations and survival benefit. Whether induction docetaxel, platinum and fluorouracil (TPF) modifies PD-L1 expression or tumour immune infiltrates is unknown. PATIENTS AND METHODS: Patients with locally advanced HNSCC treated at Gustave Roussy (Villejuif, France) between 2006 and 2013 by induction TPF followed by surgery were retrospectively considered. Patients with paired samples (pre-TPF and post-TPF) were kept for further analysis. PD-L1 expression was quantified by immunohistochemistry according to a validated protocol. The objective of the study was to compare PD-L1 expression on tumour cells (TC) and immune cells (IC) (positivity threshold of ≥5%) before and after TPF. CD8+ and Foxp3+ lymphocytes densities before and after TPF were also quantified. RESULTS: Out of 313 patients receiving induction TPF, 86 underwent surgery; paired samples were available for 21 of them. Baseline PD-L1 expression was ≥5% in two and five samples for TC and IC, respectively. A significant increase of PD-L1 expression was observed after TPF, with 15 samples (71%) presenting a positive staining in IC after induction chemotherapy (P=0.003; Wilcoxon rank-sum test) and eight samples (38%) in TC (P=0.005; Wilcoxon rank-sum test). Tumour-infiltrating CD8+ mean densities also significantly increased post-TPF (P=0.01). There was no significant difference in Foxp3+ expression, CD8/Foxp3 ratio or correlation with outcome. CONCLUSION: TPF induction chemotherapy in advanced HNSCC increases PD-L1 positivity on tumour-infiltrating ICs, as well as CD8+ lymphocytes density. These results warrant independent validation on larger datasets and might help therapeutic strategy in advanced HNSCC.

The ratio of CD8+/FOXP3 T lymphocytes infiltrating breast tissues predicts the relapse of ductal carcinoma in situ.

In a series of 248 tumor samples obtained from image-guided biopsies from patients diagnosed with ductal carcinoma in situ of the breast, we attempted to identify biomarkers that predict microinfiltration at definitive surgery or relapse during follow-up. For this, we used immunohistochemical methods, followed by automated image analyses, to measure the mean diameter of nuclei (which correlates with ploidy), the phosphorylation of eukaryotic initiation factor 2α (eIF2α, which reflects endoplasmic reticulum stress) as well as the density and ratio of CD8+ cytotoxic T lymphocytes and FOXP3+ regulatory T cells. The median nuclear diameter of malignant cells correlated with eIF2α phosphorylation (in cancerous tissue), which in turn correlated with the density of the CD8+ infiltrate and the CD8+/FOXP3 ratio (both in cancerous and the adjacent non-cancerous parenchyma). Neither microinfiltration nor lymph node involvement was associated with the probability of relapse. Both correlated positively with the CD8+/FOXP3 ratio in the malignant area. In contrast, relapse was associated with a paucity of the CD8+ infiltrate as well as an unfavorable CD8+/FOXP3 ratio, both in malignant and non-malignant parenchyma. The combined analysis of the CD8+/FOXP3 ratio in cancerous and non-cancerous tissues revealed a significant impact of their interaction on the probability of relapse, but not on the presence of microinfiltration or lymph node metastasis. Altogether, these results support the idea of an immunosurveillance system that determines the risk of relapse in ductal carcinoma in situ of the breast.

Analyses of nuclear proteins and nucleic acid structures using atomic force microscopy.

Since the inception of atomic force microscopy (AFM) in 1986, the value of this technology for exploring the structure and biophysical properties of a variety of biological samples has been increasingly recognized. AFM provides the opportunity to both image samples at nanometer resolution and also measure the forces on the surface of the sample. Here, we describe a variety of methods for studying nuclear samples including single nucleic acid molecules, higher-order chromatin structures, the nucleolus, and the nucleus. Protocols to prepare nucleic acids, nucleic acid-protein complexes, reconstituted chromatin, the cell nucleus, and the nucleolus are included, as well as protocols describing how to prepare the AFM substrate and the AFM tip. Finally, we describe how to perform conventional imaging, high-speed imaging, recognition imaging, force spectroscopy, and nanoindentation experiments.

Synergy of Radiotherapy and a Cancer Vaccine for the Treatment of HPV-Associated Head and Neck Cancer.

There is growing interest in the association of radiotherapy and immunotherapy for the treatment of solid tumors. Here, we report an extremely effective combination of local irradiation (IR) and Shiga Toxin B (STxB)-based human papillomavirus (HPV) vaccination for the treatment of HPV-associated head and neck squamous cell carcinoma (HNSCC). The efficacy of the irradiation and vaccine association was tested using a model of HNSCC obtained by grafting TC-1/luciferase cells at a submucosal site of the inner lip of immunocompetent mice. Irradiation and the STxB-E7 vaccine acted synergistically with both single and fractionated irradiation schemes, resulting in complete tumor clearance in the majority of the treated mice. A dose threshold of 7.5 Gy was required to elicit the dramatic antitumor response. The combined treatment induced high levels of tumor-infiltrating, antigen-specific CD8(+) T cells, which were required to trigger the antitumor activity. Treatment with STxB-E7 and irradiation induced CD8(+) T-cell memory, which was sufficient to exert complete antitumor responses in both local recurrences and distant metastases. We also report for the first time that a combination therapy based on local irradiation and vaccination induces an increased pericyte coverage (as shown by αSMA and NG2 staining) and ICAM-1 expression on vessels. This was associated with enhanced intratumor vascular permeability that correlated with the antitumor response, suggesting that the combination therapy could also act through an increased accessibility for immune cells. The combination strategy proposed here offers a promising approach that could potentially be transferred into early-phase clinical trials.

[The nucleolus: structure, functions, amd associated diseases]. / Le nucléole: structure, fonctions et maladies associées.

In eukaryotes, the nucleolus is the ribosome factory. The nucleolus is a very active large nuclear domain resulting from the equilibrium between level of ribosomal gene transcription, efficiency of rRNA processing and transport of the ribosomal subunits (40S and 60S) towards the cytoplasm. The ribosome production is regulated and is linked with cell growth and cell proliferation. The ribosome production is stopped during mitosis but the nucleolar machineries are inherited in daughter cells and the nucleolar reassembly is a very early event at the exit of mitosis. The nucleolus is also a multifunctional domain involved in nuclear architecture and specific interaction with some nuclear bodies. Finally, several human diseases appear to result from mutations of nucleolar proteins.

Time-lapse, photoactivation, and photobleaching imaging of nucleolar assembly after mitosis.

Nucleolus assembly starts in telophase with the benefit of building blocks passing through mitosis and lasts until cytokinesis generating the two independent interphasic cells. Several approaches make it possible to follow the dynamics of fluorescent molecules in live cells. Here, three complementary approaches are described to measure the dynamics of proteins during nucleolar assembly after mitosis: (1) rapid two-color 4-D imaging time-lapse microscopy that demonstrates the relative localization and movement of two proteins, (2) photoactivation that reveals the directionality of migration from the activated area, and (3) fluorescence recovery after photobleaching (FRAP) that measures the renewing of proteins in the bleached area. We demonstrate that the order of recruitment of the processing machineries into nucleoli results from differential sorting of intermediate structures assembled during telophase, the prenucleolar bodies.

In vivo run-on assays to monitor nascent precursor RNA transcripts.

Biochemical methods have provided mechanistic insights into the different transcription phases during which the RNA polymerase is assembled at gene promoter and becomes engaged in the elongation of nascent transcripts. Evidence that transcription takes place in specific regions of the nucleus has fuelled the need to develop assays that can be performed in living cells and provide information on the location of the specific foci, where transcription takes place. In this chapter, we describe a method that is based on the incorporation of a fluorine-conjugated uridine analogue, incorporation that can be monitored by immunofluorescence and light microscopy using specific fluorochrome-conjugated monoclonal antibodies. This assay allows direct monitoring of active transcription foci in living cells. When coupled to suitable software, the method outlined here also provides a semiquantitative approach to measure the number of active transcription foci that correlate with the proliferation state of the cell. Therefore, the assay we present here is a sensitive analytical tool to monitor the topology of transcription foci in the eukaryotic cell nucleus and to gain insight into transcription rates.

The chromatin remodelling complex B-WICH changes the chromatin structure and recruits histone acetyl-transferases to active rRNA genes.

The chromatin remodelling complex B-WICH, which comprises the William syndrome transcription factor (WSTF), SNF2h, and nuclear myosin 1 (NM1), is involved in regulating rDNA transcription, and SiRNA silencing of WSTF leads to a reduced level of 45S pre-rRNA. The mechanism behind the action of B-WICH is unclear. Here, we show that the B-WICH complex affects the chromatin structure and that silencing of the WSTF protein results in a compaction of the chromatin structure over a 200 basepair region at the rRNA promoter. WSTF knock down does not show an effect on the binding of the rRNA-specific enhancer and chromatin protein UBF, which contributes to the chromatin structure at active genes. Instead, WSTF knock down results in a reduced level of acetylated H3-Ac, in particular H3K9-Ac, at the promoter and along the gene. The association of the histone acetyl-transferases PCAF, p300 and GCN5 with the promoter is reduced in WSTF knock down cells, whereas the association of the histone acetyl-transferase MOF is retained. A low level of H3-Ac was also found in growing cells, but here histone acetyl-transferases were present at the rDNA promoter. We propose that the B-WICH complex remodels the chromatin structure at actively transcribed rRNA genes, and this allows for the association of specific histone acetyl-transferases.

The traffic of proteins between nucleolar organizer regions and prenucleolar bodies governs the assembly of the nucleolus at exit of mitosis.

The building of nuclear bodies after mitosis is a coordinated event crucial for nuclear organization and function. The nucleolus is assembled during early G(1) phase. Here, two periods (early G1a and early G1b) have been defined. During these periods, the nucleolar compartments (DFC, GC) corresponding to different steps of ribosome biogenesis are progressively assembled. In telophase, rDNA transcription is first activated and PNBs (reservoirs of nucleolar processing proteins) are formed. The traffic of the processing proteins between incipient nucleoli and PNBs was analyzed using photoactivation. We demonstrate that the DFC protein fibrillarin passes from one incipient nucleolus to other nucleoli but not to PNBs, and that the GC proteins, B23/NPM and Nop52, shuttle between PNBs and incipient nucleoli. This difference in traffic suggests a way of regulating assembly first of DFC and then of GC. The time of residency of GC proteins is high in incipient nucleoli compared to interphase nuclei, it decreases in LMB-treated early G1a cells impairing the assembly of GC. Because the assembly of the nucleolus and that of the Cajal body at the exit from mitosis are both sensitive to CRM1 activity, we discuss the fact that assembly of GC and/or its interaction with DFC in early G1a depends on shuttling between PNBs and NORs in a manner dependent on Cajal body assembly.

Transcriptional control of gene expression by actin and myosin.

Recent years have witnessed a new turn in the field of gene expression regulation. Actin and an ever-growing family of actin-associated proteins have been accepted as members of the nuclear crew, regulating eukaryotic gene transcription. In complex with heterogeneous nuclear ribonucleoproteins and certain myosin species, actin has been shown to be an important regulator in RNA polymerase II transcription. Furthermore, actin-based molecular motors are believed to facilitate RNA polymerase I transcription and possibly downstream events during rRNA biogenesis. Probably these findings represent the tip of the iceberg of a rapidly expanding area within the functional architecture of the cell nucleus. Further studies will contribute to clarify how actin mediates nuclear functions with a glance to cytoplasmic signalling. These discoveries have the potential to define novel regulatory networks required to control gene expression at multiple levels.

Time-lapse microscopy and fluorescence resonance energy transfer to analyze the dynamics and interactions of nucleolar proteins in living cells.

The dynamics of proteins play a key role in the organization and control of nuclear functions. Techniques were developed recently to observe the movement and interactions of proteins in living cells; time-lapse microscopy using fluorescent-tagged proteins gives access to observations of nuclear protein trafficking over time, and fluorescence resonance energy transfer (FRET) is used to investigate protein interactions in the time-lapse mode. In this chapter, we describe the application of these two approaches to follow the recruitment of nucleolar processing proteins at the time of nucleolar assembly. We question the role of prenucleolar bodies (PNB) during migration of the processing proteins from the chromosome periphery to sites of ribosomal genes (rDNA) transcription. The order of recruitment of different processing proteins into nucleoli is the consequence of differential sorting from the same PNBs. The dynamics of the interactions between processing proteins in PNBs suggest that PNBs are preassembly platforms for ribosomal RNA (rRNA) processing complexes.