Søren Lykke-Andersen

RNA Exosome Depletion Reveals Transcription Upstream of Active Human Promoters

Studies have shown that the bulk of eukaryotic genomes is transcribed. Transcriptome maps are frequently updated, but low-abundant transcripts have probably gone unnoticed. To eliminate RNA degradation, we depleted the exonucleolytic RNA exosome from human cells and then subjected the RNA to tiling microarray analysis. This revealed a class of short, polyadenylated and highly unstable RNAs. These promoter upstream transcripts (PROMPTs) are produced ∼0.5 to 2.5 kilobases upstream of active transcription start sites. PROMPT transcription occurs in both sense and antisense directions with respect to the downstream gene. In addition, it requires the presence of the gene promoter and is positively correlated with gene activity. We propose that PROMPT transcription is a common characteristic of RNA polymerase II (RNAPII) transcribed genes with a possible regulatory potential.

SMG6 promotes endonucleolytic cleavage of nonsense mRNA in human cells

From yeast to humans, mRNAs harboring premature termination codons (PTCs) are recognized and degraded by nonsense-mediated mRNA decay (NMD). However, degradation mechanisms of NMD have been suggested to differ between species. In Drosophila melanogaster, NMD is initiated by endonucleolysis near the PTC, whereas in yeast and human cells the current view posits that NMD occurs by exonucleolysis from one or both RNA termini. Here we report that degradation of human nonsense mRNAs can be initiated by PTC-proximal endonucleolytic cleavage. We identify the metazoan-specific NMD factor SMG6 as the responsible endonuclease by demonstrating that mutation of conserved residues in its nuclease domain—the C-terminal PIN motif—abolishes endonucleolysis in vivo and in vitro. Our data lead to a revised mechanistic model for degradation of nonsense mRNA in human cells and suggest that endonucleolytic cleavage is a conserved feature in metazoan NMD.

Nonsense-mediated mRNA decay: an intricate machinery that shapes transcriptomes

Nonsense-mediated mRNA decay (NMD) is probably the best characterized eukaryotic RNA degradation pathway. Through intricate steps, a set of NMD factors recognize and degrade mRNAs with translation termination codons that are positioned in abnormal contexts. However, NMD is not only part of a general cellular quality control system that prevents the production of aberrant proteins. Mammalian cells also depend on NMD to dynamically adjust their transcriptomes and their proteomes to varying physiological conditions. In this Review, we discuss how NMD targets mRNAs, the types of mRNAs that are targeted, and the roles of NMD in cellular stress, differentiation and maturation processes.

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.

The human core exosome interacts with differentially localized processive RNases: hDIS3 and hDIS3L

The eukaryotic RNA exosome is a ribonucleolytic complex involved in RNA processing and turnover. It consists of a nine‐subunit catalytically inert core that serves a structural function and participates in substrate recognition. Best defined in Saccharomyces cerevisiae, enzymatic activity comes from the associated subunits Dis3p (Rrp44p) and Rrp6p. The former is a nuclear and cytoplasmic RNase II/R‐like enzyme, which possesses both processive exo‐ and endonuclease activities, whereas the latter is a distributive RNase D‐like nuclear exonuclease. Although the exosome core is highly conserved, identity and arrangements of its catalytic subunits in different vertebrates remain elusive. Here, we demonstrate the association of two different Dis3p homologs—hDIS3 and hDIS3L—with the human exosome core. Interestingly, these factors display markedly different intracellular localizations: hDIS3 is mainly nuclear, whereas hDIS3L is strictly cytoplasmic. This compartmental distribution reflects the substrate preferences of the complex in vivo. Both hDIS3 and hDIS3L are active exonucleases; however, only hDIS3 has retained endonucleolytic activity. Our data suggest that three different ribonucleases can serve as catalytic subunits for the exosome in human cells.

Origins and activities of the eukaryotic exosome.

The exosome is a multi-subunit 3′-5′ exonucleolytic complex that is conserved in structure and function in all eukaryotes studied to date. The complex is present in both the nucleus and cytoplasm, where it continuously works to ensure adequate quantities and quality of RNAs by facilitating normal RNA processing and turnover, as well as by participating in more complex RNA quality-control mechanisms. Recent progress in the field has convincingly shown that the nucleolytic activity of the exosome is maintained by only two exonuclease co-factors, one of which is also an endonuclease. The additional association of the exosome with RNA-helicase and poly(A) polymerase activities results in a flexible molecular machine that is capable of dealing with the multitude of cellular RNA substrates that are found in eukaryotic cells. Interestingly, the same basic set of enzymatic activities is found in prokaryotic cells, which might therefore illustrate the evolutionary origin of the eukaryotic system. In this Commentary, we compare the structural and functional characteristics of the eukaryotic and prokaryotic RNA-degradation systems, with an emphasis on some of the functional networks in which the RNA exosome participates in eukaryotes.

The eukaryotic RNA exosome: Same scaffold but variable catalytic subunits

The RNA exosome is a versatile ribonucleolytic protein complex that participates in a multitude of cellular RNA processing and degradation events. It consists of an invariable nine-subunit core that associates with a variety of enzymatically active subunits and co-factors. These contribute to or even provide the catalytic activity and substrate specificity of the complex. The S. cerevisiae exosome has been intensively studied since its discovery in 1997 and thus serves as the archetype of eukaryotic exosomes. Notably, its catalytic potential, derived exclusively from associated subunits, differs between the nuclear and cytoplasmic versions of the complex. The same holds true for other eukaryotes, however, recent discoveries from various laboratories including our own have revealed that there are variations on this theme. Here, we review the latest findings concerning catalytic subunits of eukaryotic exosomes, and we discuss the apparent need for differential composition and subcellular distribution of exosome variants.

Ratjadones inhibit nuclear export by blocking CRM1/exportin 1.

In addition to previously isolated ratjadone A we describe three new members of this family, ratjadones B, C, and D, from another strain of the myxobacterium Sorangium cellulosum. We have investigated the properties of these ratjadones with respect to their activity on mammalian cell lines. We found IC(50) values in the picomolar range and a significant increase in the size of nuclei. A further examination showed that they inhibit the export of the leucine-rich nuclear export signal (LR-NES) containing proteins in different cell lines. Ratjadones are able to inhibit the formation of the nuclear export complex composed of the CRM1, RanGTP, and the cargo protein, as shown by two different in vitro assays. Finally, the binding of ratjadone C to CRM1 was demonstrated. These ratjadone activities are in the same concentration range as described for the polyketide leptomycin B (LMB) from Streptomyces sp. Like LMB, it seems that the ratjadones covalently bind to CRM1, inhibit cargo protein binding via LR-NES, and thereby block nuclear export. Thus, the ratjadones represent a new class of natural compounds which inhibit proliferation in eukaryotes by blocking nuclear export.

Assembly of an Export-Competent mRNP Is Needed for Efficient Release of the 3-End Processing Complex after Polyadenylation

ABSTRACT Before polyadenylated mRNA is exported from the nucleus, the 3′-end processing complex is removed by a poorly described mechanism. In this study, we asked whether factors involved in mRNP maturation and export are also required for disassembly of the cleavage and polyadenylation complex. An RNA immunoprecipitation assay monitoring the amount of the cleavage factor (CF) IA component Rna15p associated with poly(A)+ RNA reveals defective removal of Rna15p in mutants of the nuclear export receptor Mex67p as well as other factors important for assembly of an export-competent mRNP. In contrast, Rna15p is not retained in mutants of export factors that function primarily on the cytoplasmic side of the nuclear pore. Consistent with a functional interaction between Mex67p and the 3′-end processing complex, a mex67 mutant accumulates unprocessed SSA4 transcripts and exhibits a severe growth defect when this mutation is combined with mutation of Rna15p or another CF IA subunit, Rna14p. RNAs that become processed in a mex67 mutant have longer poly(A) tails both in vivo and in vitro. This influence of Mex67p on 3′-end processing is conserved, as depletion of its human homolog, TAP/NXF1, triggers mRNA hyperadenylation. Our results indicate a function for nuclear mRNP assembly factors in releasing the 3′-end processing complex once polyadenylation is complete.

The RNA Exosome Component hRrp6 Is a Target for 5-Fluorouracil in Human Cells

The drug 5-fluorouracil (5-FU) is a widely used chemotherapeutic in the treatment of solid tumors. Recently, the essential 3′-5′ exonucleolytic multisubunit RNA exosome was implicated as a target for 5-FU in yeast. Here, we show that this is also the case in human cells. HeLa cells depleted of the inessential exosome component hRrp6, also called PM/Scl100, are significantly growth impaired relative to control cells after 5-FU administration. The selective stabilization of bona fide hRrp6 RNA substrates on 5-FU treatment suggests that this exosome component is specifically targeted. Consistently, levels of hRrp6 substrates are increased in two 5-FU–sensitive cell lines. Interestingly, whereas down-regulation of all tested core exosome components results in decreased hRrp6 levels, depletion of hRrp6 leaves levels of other exosome components unchanged. Taken together, our data position hRrp6 as a promising target for antiproliferative intervention. (Mol Cancer Res 2008;6(6):990–5)