Nop53p interacts with 5.8S rRNA co-transcriptionally, and regulates processing of pre-rRNA by the exosome

In eukaryotes, pre-rRNA processing depends on a large number of nonribosomal trans-acting factors that form intriguingly organized complexes. One of the early stages of pre-rRNA processing includes formation of the two intermediate complexes pre-40S and pre-60S, which then form the mature ribosome subunits. Each of these complexes contains specific pre-rRNAs, ribosomal proteins and processing factors. The yeast nucleolar protein Nop53p has previously been identified in the pre-60S complex and shown to affect pre-rRNA processing by directly binding to 5.8S rRNA, and to interact with Nop17p and Nip7p, which are also involved in this process. Here we show that Nop53p binds 5.8S rRNA co-transcriptionally through its N-terminal region, and that this protein portion can also partially complement growth of the conditional mutant strain Delta nop53/GAL:NOP53. Nop53p interacts with Rrp6p and activates the exosome in vitro. These results indicate that Nop53p may recruit the exosome to 7S pre-rRNA for processing. Consistent with this observation and similar to the observed in exosome mutants, depletion of Nop53p leads to accumulation of polyadenylated pre-rRNAs.

Insights into the mechanism of progressive RNA degradation by the archaeal exosome

Initially identified in yeast, the exosome has emerged as a central component of the RNA maturation and degradation machinery both in Archaea and eukaryotes. Here we describe a series of high-resolution structures of the RNase PH ring from the Pyrococcus abyssi exosome, one of them containing three 10-mer RNA strands within the exosome catalytic chamber, and report additional nucleotide interactions involving positions N5 and N7. Residues from all three Rrp41-Rrp42 heterodimers interact with a single RNA molecule, providing evidence for the functional relevance of exosome ring-like assembly in RNA processivity. Furthermore, an ADP-bound structure showed a rearrangement of nucleotide interactions at site N1, suggesting a rationale for the elimination of nucleoside diphosphate after catalysis. In combination with RNA degradation assays performed with mutants of key amino acid residues, the structural data presented here provide support for a model of exosome-mediated RNA degradation that integrates the events involving catalytic cleavage, product elimination, and RNA translocation. Finally, comparisons between the archaeal and human exosome structures provide a possible explanation for the eukaryotic exosome inability to catalyze phosphate-dependent RNA degradation.

Identification of archaeal proteins that affect the exosome function in vitro

Abstract Background The archaeal exosome is formed by a hexameric RNase PH ring and three RNA binding subunits and has been shown to bind and degrade RNA in vitro. Despite extensive studies on the eukaryotic exosome and on the proteins interacting with this complex, little information is yet available on the identification and function of archaeal exosome regulatory factors. Results Here, we show that the proteins PaSBDS and PaNip7, which bind preferentially to poly-A and AU-rich RNAs, respectively, affect the Pyrococcus abyssi exosome activity in vitro. PaSBDS inhibits slightly degradation of a poly-rA substrate, while PaNip7 strongly inhibits the degradation of poly-A and poly-AU by the exosome. The exosome inhibition by PaNip7 appears to depend at least partially on its interaction with RNA, since mutants of PaNip7 that no longer bind RNA, inhibit the exosome less strongly. We also show that FITC-labeled PaNip7 associates with the exosome in the absence of substrate RNA. Conclusions Given the high structural homology between the archaeal and eukaryotic proteins, the effect of archaeal Nip7 and SBDS on the exosome provides a model for an evolutionarily conserved exosome control mechanism.

Caspase-mediated cleavage of the exosome subunit PM/Scl-75 during apoptosis

Recent studies have implicated the dying cell as a potential reservoir of modified autoantigens that might initiate and drive systemic autoimmunity in susceptible hosts. A number of subunits of the exosome, a complex of 3'→5' exoribonucleases that functions in a variety of cellular processes, are recognized by the so-called anti-PM/Scl autoantibodies, found predominantly in patients suffering from an overlap syndrome of myositis and scleroderma. Here we show that one of these subunits, PM/Scl-75, is cleaved during apoptosis. PM/Scl-75 cleavage is inhibited by several different caspase inhibitors. The analysis of PM/Scl-75 cleavage by recombinant caspase proteins shows that PM/Scl-75 is efficiently cleaved by caspase-1, to a smaller extent by caspase-8, and relatively inefficiently by caspase-3 and caspase-7. Cleavage of the PM/Scl-75 protein occurs in the C-terminal part of the protein at Asp369 (IILD369↓G), and at least a fraction of the resulting N-terminal fragments of PM/Scl-75 remains associated with the exosome. Finally, the implications of PM/Scl-75 cleavage for exosome function and the generation of anti-PM/Scl-75 autoantibodies are discussed.

Endocytic trafficking and oligodendroglial exosome secretion in axon-glia communication and myelination

Oligodendrocytes form specialized plasma membrane extensions which spirally enwrap axons, thereby building up the myelin sheath. During myelination, oligodendrocytes produce large amounts of membrane components. Oligodendrocytes can be seen as a complex polarized cell type with two distinct membrane domains, the plasma membrane surrounding the cell body and the myelin membrane. SNARE proteins mediate the fusion of vesicular cargoes with their target membrane. We propose a model in which the major myelin protein PLP is transported by two different pathways. VAMP3 mediates the non-polarized transport of newly synthesized PLP via recycling endosomes to the plasma membrane, while transport of PLP from late endosomes/lysosomes to myelin is controlled by VAMP7. In the second part of the thesis, the role of exosome secretion in glia to axon signaling was studied. Further studies are required to clarify whether VAMP7 also controls exosome secretion. The thesis further focused on putative metabolic effects in the target neurons. Oligodendroglial exosomes showed no obvious influences on neuronal metabolic activity. Analysis of the phosphorylation levels of the neurofilament heavy subunit revealed a decrease in presence of oligodendrocytes, indicating effects of oligodendroglial exosomes on the neuronal cytoskeleton. Finally, candidates for kinases which are possibly activated upon influence of oligodendroglial exosomes and could influence neuronal survival were identified.

THE DOMAINS OF THE CATALYTIC SUBUNIT OF THE EUKARYOTIC RNA DEGRADING EXOSOME, RRP44P, HAVE DISTINCT FUNCTIONS

The exosome is a 3’ to 5’ exoribonuclease complex that consists of ten essential subunits. In the cytoplasm, the exosome degrades mRNA in a general mRNA turnover pathway and in several mRNA surveillance pathways. In the nucleus, the exosome processes RNA precursors to form small, stable, mature RNA species, including rRNA, snRNA, and snoRNA. In addition to processing these RNAs, the nuclear exosome is also involved in degrading aberrantly processed forms of these RNAs, and others, including mRNA. The 3’ to 5’ exoribonuclease activity of the exosome is contributed by the RNB domain of the only catalytically active subunit, Rrp44p, a member of the RNase II family of enzymes. In addition to the RNB domain, Rrp44p consists of three putative RNA binding domains and has an uncharacterized N-terminus, which includes a CR3 region and PIN domain. In an effort to characterize the cellular functions of the domains of Rrp44p, this study identified a second nuclease active site in the PIN domain. Specifically, the PIN domain exhibits endoribonuclease activity in vitro and is essential for exosome function. Further analysis of the nuclease activities of Rrp44p indicate a role for the exoribonuclease activity of Rrp44p in the cytoplasmic and nuclear exosome. This work has also characterized the CR3 region of Rrp44p, a region that has not yet been characterized in any other protein. This region is needed for the majority, if not all, of the cytoplasmic exosome functions as well as for interaction with the exosome. The CR3 region, along with a histidine residue in the N-terminus of Rrp44p, may coordinate a zinc atom. Preliminary evidence supports a role for this coordination in exosome function. Further investigation, however, is needed to determine the molecular dependence of the exosome on the CR3 region of Rrp44p. Despite its initial discovery thirteen years ago, the essential function of Rrp44p, and the exosome, is not yet known. The studies presented here, however, indicate that the essential function of Rrp44p and the exosome is in the nucleus and depends on the nuclease activities of Rrp44p.

The Role of the Arched Helicases in Exosome-Mediated Function

RNA processing and degradation are two important functions that control gene expression and promote RNA fidelity in the cell. A major ribonuclease complex, called the exosome, is involved in both of these processes. The exosome is composed of ten essential proteins with only one catalytically active subunit, called Rrp44. While the same ten essential subunits make up both the nuclear and cytoplasmic exosome, there are nuclear and cytoplasmic exosome cofactors that promote specific exosome functions in each of the cell compartments. To date, it is unclear how the exosome distinguishes between RNA substrates. We hypothesize that compartment specific cofactors may promote the substrate specificity of the exosome. In this work, I characterize several cofactors of the exosome, both nuclear and cytoplasmic. First, I describe the arch domain, which is a unique domain in a nuclear and a cytoplasmic cofactor of the exosome. Specifically, I show that the arch domain of the nuclear exosome cofactor, Mtr4, is required for specific exosome-mediated activities and overlaps functionally with the exosome-associated exonuclease, Rrp6. Further, I show that the arch domain of Ski2 is required for the degradation of normal and aberrant mRNAs. Additionally, this work describes in detail the Mtr4 domains involved in the physical association with other RNA processing proteins. Further, I characterize the minimal Mtr4-binding region in a third exosome cofactor, Trf5. Understanding how exosome cofactors synergistically promote exosome function will provide us a better understanding of how the exosome complex precisely regulates its catalytic activities. As described here, cofactors play a major role in determining the substrate specificity of the nuclear and cytoplasmic exosome. Moreover, specific accessory domains, which are not involved in the catalytic function of the cofactor, are required for substrate targeting of the eukaryotic RNA exosome.

Exosomes in prostate cancer: Putting together the pieces of a puzzle

Exosomes have been shown to act as mediators for cell to cell communication and as a potential source of biomarkers for many diseases, including prostate cancer. Exosomes are nanosized vesicles secreted by cells and consist of proteins normally found in multivesicular bodies, RNA, DNA and lipids. As a potential source of biomarkers, exosomes have attracted considerable attention, as their protein content resembles that of their cells of origin, even though it is noted that the proteins, miRNAs and lipids found in the exosomes are not a reflective stoichiometric sampling of the contents from the parent cells. While the biogenesis of exosomes in dendritic cells and platelets has been extensively characterized, much less is known about the biogenesis of exosomes in cancer cells. An understanding of the processes involved in prostate cancer will help to further elucidate the role of exosomes and other extracellular vesicles in prostate cancer progression and metastasis. There are few methodologies available for general isolation of exosomes, however validation of those methodologies is necessary to study the role of exosomal-derived biomarkers in various diseases. In this review, we discuss “exosomes” as a member of the family of extracellular vesicles and their potential to provide candidate biomarkers for prostate cancer.

Functions and therapeutic roles of exosomes in cancer

The role of exosomes in cancer development has become the focus of much research, due to the many emerging roles possessed by exosomes. These micro-vesicles that are ubiquitously released in to the extracellular milieu, have been found to regulate immune system function, particularly in tumorigenesis, as well as conditioning future metastatic sites for the attachment and growth of tumor tissue. Through an interaction with a range of host tissue, exosomes are able to generate a pro-tumor environment that is essential for carcinogenesis. Herein, we discuss the contents of exosomes and their contribution to tumorigenesis, as well as their role in chemotherapeutic resistance and the development of novel cancer treatments and the identification of cancer biomarkers.

Examination of EXOSC4 as a new prognostic marker and a novel therapeutic avenue in lung adenocarcinoma

Although prevention and early detection of the disease greatly improved over the past few years, lung cancer remains the leading cause of cancer deaths. In order to be able to treat a larger population, we are in urgent need for novel treatments. While it is known that DNA repair genes play a major role in the oncogenic transformation, they also represent a weakness of cancers that constitute a therapeutic opportunity. To identify novel DNA repair genes implicated in Lung cancers, we conducted an in silico investigation to identify genes co-regulated with two DNA repair factors, BRCA2 and hSSB1. This approach allowed for the identification of EXOSC4, a component of the RNA Exosome machinery, as a potential factor involved in the maintenance of genome stability and that is deregulated in lung cancer.

Label-free detection of single biological molecules using microtoroid optical resonators

Being able to detect a single molecule without the use of labels has been a long standing goal of bioengineers and physicists. This would simplify applications ranging from single molecular binding studies to those involving public health and security, improved drug screening, medical diagnostics, and genome sequencing. One promising technique that has the potential to detect single molecules is the microtoroid optical resonator. The main obstacle to detecting single molecules, however, is decreasing the noise level of the measurements such that a single molecule can be distinguished from background. We have used laser frequency locking in combination with balanced detection and data processing techniques to reduce the noise level of these devices and report the detection of a wide range of nanoscale objects ranging from nanoparticles with radii from 100 to 2.5 nm, to exosomes, ribosomes, and single protein molecules (mouse immunoglobulin G and human interleukin-2). We further extend the exosome results towards creating a non-invasive tumor biopsy assay. Our results, covering several orders of magnitude of particle radius (100 nm to 2 nm), agree with the `reactive' model prediction for the frequency shift of the resonator upon particle binding. In addition, we demonstrate that molecular weight may be estimated from the frequency shift through a simple formula, thus providing a basis for an ``optical mass spectrometer'' in solution. We anticipate that our results will enable many applications, including more sensitive medical diagnostics and fundamental studies of single receptor-ligand and protein-protein interactions in real time. The thesis summarizes what we have achieved thus far and shows that the goal of detecting a single molecule without the use of labels can now be realized.

The extracellular vesicles of the helminth pathogen, Fasciola hepatica: biogenesis pathways and cargo molecules involved in parasite pathogenesis.

Extracellular vesicles (EVs) released by parasites have important roles in establishing and maintaining infection. Analysis of the soluble and vesicular secretions of adult Fasciola hepatica has established a definitive characterisation of the total secretome of this zoonotic parasite. Fasciola secretes at least two sub-populations of EVs that differ according to size, cargo molecules and site of release from the parasite. The larger EVs are released from the specialised cells that line the parasite gastrodermus and contain the zymogen of the 37 kDa cathepsin L peptidase that performs a digestive function. The smaller exosome-like vesicle population originate from multivesicular bodies within the tegumental syncytium and carry many previously described immunomodulatory molecules that could be delivered into host cells. By integrating our proteomics data with recently available transcriptomic datasets we have detailed the pathways involved with EV biogenesis in F. hepatica and propose that the small exosome biogenesis occurs via ESCRT-dependent MVB formation in the tegumental syncytium before being shed from the apical plasma membrane. Furthermore, we found that the molecular machinery required for EV biogenesis is constitutively expressed across the intra-mammalian development stages of the parasite. By contrast, the cargo molecules packaged within the EVs are developmentally regulated, most likely to facilitate the parasites migration through host tissue and to counteract host immune attack.

Human factors required for mycobacterium tuberculosis macrophage infection

Tese de doutoramento, Farmácia (Biologia Celular e Molecular), Universidade de Lisboa, Faculdade de Farmácia, 2014