Michal Lubas

Interaction Profiling Identifies the Human Nuclear Exosome Targeting Complex

The RNA exosome is a conserved degradation machinery, which obtains full activity only when associated with cofactors. The most prominent activator of the yeast nuclear exosome is the RNA helicase Mtr4p, acting in the context of the Trf4p/Air2p/Mtr4p polyadenylation (TRAMP) complex. The existence of a similar activator(s) in humans remains elusive. By establishing an interaction network of the human nuclear exosome, we identify the trimeric Nuclear Exosome Targeting (NEXT) complex, containing hMTR4, the Zn-knuckle protein ZCCHC8, and the putative RNA binding protein RBM7. ZCCHC8 and RBM7 are excluded from nucleoli, and consistently NEXT is specifically required for the exosomal degradation of promoter upstream transcripts (PROMPTs). We also detect putative homolog TRAMP subunits hTRF4-2 (Trf4p) and ZCCHC7 (Air2p) in hRRP6 and hMTR4 precipitates. However, at least ZCCHC7 function is restricted to nucleoli. Our results suggest that human nuclear exosome degradation pathways comprise modules of spatially organized cofactors that diverge from the yeast model.

RNA decay machines: The exosome ☆

The multisubunit RNA exosome complex is a major ribonuclease of eukaryotic cells that participates in the processing, quality control and degradation of virtually all classes of RNA in Eukaryota. All this is achieved by about a dozen proteins with only three ribonuclease activities between them. At first glance, the versatility of the pathways involving the exosome and the sheer multitude of its substrates are astounding. However, after fifteen years of research we have some understanding of how exosome activity is controlled and applied inside the cell. The catalytic properties of the eukaryotic exosome are fairly well described and attention is now drawn to how the interplay between these activities impacts cell physiology. Also, it has become evident that exosome function relies on many auxiliary factors, which are intensely studied themselves. In this way, the focus of exosome research is slowly leaving the test tube and moving back into the cell. The exosome also has an interesting evolutionary history, which is evident within the eukaryotic lineage but only fully appreciated when considering similar protein complexes found in Bacteria and Archaea. Thus, while we keep this review focused on the most comprehensively described yeast and human exosomes, we shall point out similarities or dissimilarities to prokaryotic complexes and proteins where appropriate. The article is divided into three parts. In Part One we describe how the exosome is built and how it manifests in cells of different organisms. In Part Two we detail the enzymatic properties of the exosome, especially recent data obtained for holocomplexes. Finally, Part Three presents an overview of the RNA metabolism pathways that involve the exosome. This article is part of a Special Issue entitled: RNA Decay mechanisms.

Exonuclease hDIS3L2 specifies an exosome-independent 3′-5′ degradation pathway of human cytoplasmic mRNA

Turnover of mRNA in the cytoplasm of human cells is thought to be redundantly conducted by the monomeric 5′‐3′ exoribonuclease hXRN1 and the 3′‐5′ exoribonucleolytic RNA exosome complex. However, in addition to the exosome‐associated 3′‐5′ exonucleases hDIS3 and hDIS3L, the human genome encodes another RNase II/R domain protein—hDIS3L2. Here, we show that hDIS3L2 is an exosome‐independent cytoplasmic mRNA 3′‐5′ exonuclease, which exhibits processive activity on structured RNA substrates in vitro. hDIS3L2 associates with hXRN1 in an RNA‐dependent manner and can, like hXRN1, be found on polysomes. The impact of hDIS3L2 on cytoplasmic RNA metabolism is revealed by an increase in levels of cytoplasmic RNA processing bodies (P‐bodies) upon hDIS3L2 depletion, which also increases half‐lives of investigated mRNAs. Consistently, RNA sequencing (RNA‐seq) analyses demonstrate that depletion of hDIS3L2, like downregulation of hXRN1 and hDIS3L, causes changed levels of multiple mRNAs. We suggest that hDIS3L2 is a key exosome‐independent effector of cytoplasmic mRNA metabolism.

DMR1 ( CCM1/ YGR150C) of Saccharomyces cerevisiae Encodes an RNA-Binding Protein from the Pentatricopeptide Repeat Family Required for the Maintenance of the Mitochondrial 15S Ribosomal RNA

Pentatricopeptide repeat (PPR) proteins form the largest known RNA-binding protein family and are found in all eukaryotes, being particularly abundant in higher plants. PPR proteins localize mostly in mitochondria and chloroplasts, where they modulate organellar genome expression on the post-transcriptional level. The Saccharomyces cerevisiae DMR1 (CCM1, YGR150C) encodes a PPR protein that localizes to mitochondria. Deletion of DMR1 results in a complete and irreversible loss of respiratory capacity and loss of wild-type mtDNA by conversion to ρ−/ρ0 petites, regardless of the presence of introns in mtDNA. The phenotype of the dmr1Δ mitochondria is characterized by fragmentation of the small subunit mitochondrial rRNA (15S rRNA), that can be reversed by wild-type Dmr1p. Other mitochondrial transcripts, including the large subunit mitochondrial rRNA (21S rRNA), are not affected by the lack of Dmr1p. The purified Dmr1 protein specifically binds to different regions of 15S rRNA in vitro, consistent with the deletion phenotype. Dmr1p is therefore the first yeast PPR protein, which has an rRNA target and is probably involved in the biogenesis of mitochondrial ribosomes and translation.

Principles for RNA metabolism and alternative transcription initiation within closely spaced promoters

Mammalian transcriptomes are complex and formed by extensive promoter activity. In addition, gene promoters are largely divergent and initiate transcription of reverse-oriented promoter upstream transcripts (PROMPTs). Although PROMPTs are commonly terminated early, influenced by polyadenylation sites, promoters often cluster so that the divergent activity of one might impact another. Here we found that the distance between promoters strongly correlates with the expression, stability and length of their associated PROMPTs. Adjacent promoters driving divergent mRNA transcription support PROMPT formation, but owing to polyadenylation site constraints, these transcripts tend to spread into the neighboring mRNA on the same strand. This mechanism to derive new alternative mRNA transcription start sites (TSSs) is also evident at closely spaced promoters supporting convergent mRNA transcription. We suggest that basic building blocks of divergently transcribed core promoter pairs, in combination with the wealth of TSSs in mammalian genomes, provide a framework with which evolution shapes transcriptomes.

Emerging connections between RNA and autophagy

ABSTRACT Macroautophagy/autophagy is a key catabolic process, essential for maintaining cellular homeostasis and survival through the removal and recycling of unwanted cellular material. Emerging evidence has revealed intricate connections between the RNA and autophagy research fields. While a majority of studies have focused on protein, lipid and carbohydrate catabolism via autophagy, accumulating data supports the view that several types of RNA and associated ribonucleoprotein complexes are specifically recruited to phagophores (precursors to autophagosomes) and subsequently degraded in the lysosome/vacuole. Moreover, recent studies have revealed a substantial number of novel autophagy regulators with RNA-related functions, indicating roles for RNA and associated proteins not only as cargo, but also as regulators of this process. In this review, we discuss widespread evidence of RNA catabolism via autophagy in yeast, plants and animals, reviewing the molecular mechanisms and biological importance in normal physiology, stress and disease. In addition, we explore emerging evidence of core autophagy regulation mediated by RNA-binding proteins and noncoding RNAs, and point to gaps in our current knowledge of the connection between RNA and autophagy. Finally, we discuss the pathological implications of RNA-protein aggregation, primarily in the context of neurodegenerative disease.

Mutually Exclusive CBC-Containing Complexes Contribute to RNA Fate

Summary The nuclear cap-binding complex (CBC) stimulates processing reactions of capped RNAs, including their splicing, 3′-end formation, degradation, and transport. CBC effects are particular for individual RNA families, but how such selectivity is achieved remains elusive. Here, we analyze three main CBC partners known to impact different RNA species. ARS2 stimulates 3′-end formation/transcription termination of several transcript types, ZC3H18 stimulates degradation of a diverse set of RNAs, and PHAX functions in pre-small nuclear RNA/small nucleolar RNA (pre-snRNA/snoRNA) transport. Surprisingly, these proteins all bind capped RNAs without strong preferences for given transcripts, and their steady-state binding correlates poorly with their function. Despite this, PHAX and ZC3H18 compete for CBC binding and we demonstrate that this competitive binding is functionally relevant. We further show that CBC-containing complexes are short lived in vivo, and we therefore suggest that RNA fate involves the transient formation of mutually exclusive CBC complexes, which may only be consequential at particular checkpoints during RNA biogenesis.

eIF5A is required for autophagy by mediating ATG3 translation

Autophagy is an essential catabolic process responsible for recycling of intracellular material and preserving cellular fidelity. Key to the autophagy pathway is the ubiquitin‐like conjugation system mediating lipidation of Atg8 proteins and their anchoring to autophagosomal membranes. While regulation of autophagy has been characterized at the level of transcription, protein interactions and post‐translational modifications, its translational regulation remains elusive. Here we describe a role for the conserved eukaryotic translation initiation factor 5A (eIF5A) in autophagy. Identified from a high‐throughput screen, we find that eIF5A is required for lipidation of LC3B and its paralogs and promotes autophagosome formation. This feature is evolutionarily conserved and results from the translation of the E2‐like ATG3 protein. Mechanistically, we identify an amino acid motif in ATG3 causing eIF5A dependency for its efficient translation. Our study identifies eIF5A as a key requirement for autophagosome formation and demonstrates the importance of translation in mediating efficient autophagy.

Biochemistry and Function of RNA Exosomes.

Discovery of the evolutionary conserved RNA exosome was a milestone in RNA biology. First identified as an activity essential for the processing of ribosomal RNA, the exosome has since proved to be central for RNA processing and degradation in both the nucleus and the cytoplasm of eukaryotic cells. This multisubunit protein complex consists of a catalytically inert 9-subunit core endowed with associated ribonucleolytic activities and further assisted by compartment-specific cofactors required for its activation and substrate targeting. Although many features of exosome biology are known, fundamental aspects are still under investigation. In this chapter, we review current biochemical and functional knowledge of eukaryotic exosomes. After introducing some of their nuclear and cytoplasmic functions, we discuss the structural organization and evolutionary aspects of exosome complexes. Finally, we describe catalytic properties of the complex and its regulation by cofactors.

The RNA Exosome Adaptor ZFC3H1 Functionally Competes with Nuclear Export Activity to Retain Target Transcripts

Summary Mammalian genomes are promiscuously transcribed, yielding protein-coding and non-coding products. Many transcripts are short lived due to their nuclear degradation by the ribonucleolytic RNA exosome. Here, we show that abolished nuclear exosome function causes the formation of distinct nuclear foci, containing polyadenylated (pA+) RNA secluded from nucleocytoplasmic export. We asked whether exosome co-factors could serve such nuclear retention. Co-localization studies revealed the enrichment of pA+ RNA foci with “pA-tail exosome targeting (PAXT) connection” components MTR4, ZFC3H1, and PABPN1 but no overlap with known nuclear structures such as Cajal bodies, speckles, paraspeckles, or nucleoli. Interestingly, ZFC3H1 is required for foci formation, and in its absence, selected pA+ RNAs, including coding and non-coding transcripts, are exported to the cytoplasm in a process dependent on the mRNA export factor AlyREF. Our results establish ZFC3H1 as a central nuclear pA+ RNA retention factor, counteracting nuclear export activity.