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

Abstract

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.

Figure 1

Mammalian genomes encode two proteins, Dis3 and Dis3L, with domain composition identical to that of yeast Dis3p. (A) Domain organization of RNase II/R enzymes from different species. E. coli RNase II and Dis3 proteins from yeast and humans contain an RNase II/R-like region featuring a similar modular arrangement with two cold-shock domains (CSD1 and CSD2), an RNB domain and a C-terminal S1 domain. In addition, Dis3 factors have an extended N-terminal part including the PIN domain. (B) Sequence alignment of the RNB domains of S. cerevisiae Dis3p (Dis3p_Sc), Homo sapiens hDIS3 (accession BAF92610) and hDIS3L (accession Q8TF46) (DIS3_Hs and DIS3L_Hs) and Mus musculus Dis3 and Dis3L (Dis3_Mm and Dis3l_Mm). The four conserved aspartic acid residues marked in red are involved in the coordination of magnesium in the catalytic centre. (C) Sequence alignment of the PIN domains of the five Dis3 proteins from (B). The four acidic residues essential for catalysis are marked in red. Two of these are absent from the Dis3L proteins.

Figure 2

hDIS3, but not hDIS3L, complements a Dis3p-depleted yeast strain. (A) Yeast complementation assay. LEU-marked plasmids containing hDIS3, hDIS3L, yeast DIS3 or no insert (empty vector control) were transformed into a strain harbouring endogenous DIS3 under control of a doxycycline-repressible promoter. Growth phenotypes of the resulting strains were analysed after 60 h in the absence (−LEU, endogenous DIS3 expressed) or presence (−LEU +DOX, endogenous DIS3 repressed) of doxycycline (upper part). Analogous analysis was done using plasmids encoding hDIS3-FLAG and hDIS3L-FLAG fusions (bottom part). (B) Northern blotting analysis of hDIS3 and hDIS3L transcripts from selected total RNA samples from (A). Methylene-blue staining of the membrane after transfer (top) and re-hybridization of the blot with a scR1 probe (bottom) served as loading controls. M—RiboRuler High Range RNA Ladder (Fermentas) with bands' sizes (in nucleotides) indicated. (C) hDIS3 and hDIS3L protein production from the expression plasmids used in (A). Anti-FLAG beads with bound proteins purified from extracts from yeast transformed with constructs coding for either hDIS3-FLAG or hDIS3L-FLAG, or an empty vector were subjected to SDS–PAGE analysis (top) or western blotting using anti-FLAG antibodies (bottom). M—PageRuler Prestained Protein Ladder (Fermentas) with molecular masses indicated.

Figure 3

hDIS3 and hDIS3L can copurify the human exosome core. (A) Western blotting analyses of hRRP41-FLAG (top), hDIS3-FLAG (middle) and hDIS3L-FLAG (bottom) anti-FLAG precipitations. Uninduced (−) or tetracycline-induced (+) cells were grown in SILAC media. Input and unbound fractions, as well as eluates, were probed with α-RRP41 or α-FLAG antibodies as indicated. α-UAP56 antibodies were used as a control. ^*, ^** and ^*** indicate hRRP41-FLAG, endogenous hRRP41 and hDIS3-FLAG, respectively. (B) hRRP41, hDIS3 and hDIS3L copurifying proteins identified by MS. Exosome core components and accessory factors are shown in bold. Only proteins with a SILAC ratio above background (see Materials and methods) are shown. Moreover, proteins detected by only one peptide are omitted unless they are known exosome components, in which case the MS spectra was manually validated and the protein was marked with an asterisk. In the interest of space, the list of interactors is cut after the last identified exosome component. For full data sets, including protein scores, see Supplementary Tables S1–S3).

Figure 4

Differential subcellular localization of the three human exosome complex catalytic subunits. (A) Endogenous hDIS3 is localized mainly in the nucleus with a smaller amount in the cytoplasm. HeLa cells were subjected to immunofluorescence analysis using anti-hDIS3 antibody and visualized by confocal microscopy. Nuclei were stained with Hoechst dye. (B) An hDIS3L-myc fusion protein localizes exclusively to the cytoplasm. HEK293 Flp-In T-REx cells were transiently transfected with a plasmid bearing hDIS3L-c-myc. Protein expression was induced for 12 h with doxycycline and 24 h after transfection cells were subjected to immunofluorescence staining and confocal microscopy. (C) Endogenous hRRP6 is mainly nuclear with enrichment in the nucleoli, and some cytoplasmic staining. HeLa cells were subjected to immunofluorescence using anti-hRRP6 antibodies and inspected by confocal microscopy. (D) Analysis of the distribution of different exosome subunits in subcellular fractions of HEK293 Flp-In T-REx cells. Nuclei, cytoplasm and chromatin were separated by fractionation and similar cell equivalents were subjected to SDS–PAGE followed by western blotting using the indicated antibodies. hDIS3, hRRP6 and hRRP40 display varying degrees of dual nucleo-cytoplasmic localization, whereas hDIS3L is localized exclusively in the cytoplasm. Tubulin, the U1 70K splicing factor and histone H3 serve as cytoplasmic, nuclear and chromatin markers, respectively. The nuc/cyt fractions and the chromatin fractions were run on different gels. (E) Verification of the cellular fractionation analysis of (D) by HEK293 Flp-In T-REx cell staining of endogenous hDIS3, hDIS3L, hRRP6 and hRRP40 as indicated. Cells were inspected by epifluorescence microscopy.

Figure 5

In vivo analyses of hDIS3, hDIS3L and hRRP6 substrate preferences. (A) Western blotting analysis of HeLa cells subjected to single, double or triple knockdown of hDIS3 (3), hDIS3L (3L) and hRRP6 (6) as well as single knockdown of hRRP40 (40) or treated with control siRNA, targeting eGFP (C). Anti-hDIS3, anti-hDIS3L, anti-hRRP6, anti-hRRP40 and anti-XRN1 (control) antibodies were used as indicated. (B) Northern blotting analysis of 5.8S rRNA and 3′end extended species present in total RNA samples harvested from cells (from (A)) subjected to the indicated factor depletions. The lower panel, corresponding to the lower part of the blot, shows mature 5.8S rRNA (4-min exposure) and the upper panel, corresponding to the upper part of the blot, shows 3′end extended variants (2-h exposure) each marked with an asterisk and a number. (C) RT–qPCR determination of levels of selected PROMPT (40-2, 40-13, 40-33 and 40-59) RNAs present in total RNA samples (from (A)). Histograms represent fold-change of the knockdown sample relative to the control (C) set to 1. All values were normalized internally to GAPDH RNA levels, which did not change significantly between the samples (s.d. are shown (n=3)). (D) RT–qPCR determination as in (C) of c-MYC and c-FOS RNA levels from the above-mentioned samples (A). Histograms and normalization are like in (C) (s.d. are shown (n=3)).

Figure 6

hDIS3 and hDIS3L are both 3′ → 5′ exoribonucleases. (A) NuPAGE analysis of TAP-tagged full-length hDIS3 (left panel) and hDIS3L (right panel) WT, RNB MUT and double RNB and PIN domain mutant (DM) variants affinity purified using the TAP protein A moiety. Positions of hDIS3/hDIS3L TAP fusions bound to IgG-Sepharose beads and cleaved/eluted proteins are marked with open arrows and asterisks, respectively. Parallel purification performed using cells transfected with an empty vector served as a negative control. Eluates were used in biochemical experiments shown in (B) and in Figure 7. Lane designations: EX—extract; B—IgG-resin with bound proteins; EL—elution by TEV protease cleavage; M—PageRuler Prestained Protein Ladder (Fermentas) with molecular masses indicated. (B) hDIS3 and hDIS3L display 3′ → 5′ exoribonuclease activity abolished by mutation of the RNB domain. 5′-labelled ss17-A₁₄ substrate was incubated in a buffer containing 100 μM magnesium with hDIS3^WT (left panel) or hDIS3L^WT (right panel), their RNB MUT counterparts, an ‘empty vector' control from (A) or in the absence of added protein. Full-length WT yeast Dis3p was used as a positive control. Reactions were terminated at the indicated time points followed by denaturing PAGE and phosphorimaging. Positions of 3 nt-long marker and 4–5 nt-long final degradation products, which were highly similar between S. cerevisiae Dis3p and hDIS3/hDIS3L proteins, are marked. In the case of hDIS3 (left panel), the reaction mixtures were re-run longer to better visualize the size of terminal degradation products (bottom). Final hDIS3, hDIS3L and Dis3p protein concentrations were 0.25, 0.04 and 0.2 μM, respectively.

Figure 7

hDIS3, but not hDIS3L is endowed with PIN domain-dependent endoribonucleolytic activity. (A) hDIS3^{RNB MUT} displays endonuclease activity, which is abolished by PIN domain mutation. 5′-labelled (left), 3′-labelled (middle) or circularized (right) ss17-A₁₄ RNA substrates were incubated with hDIS3^{RNB MUT}, hDIS3^DM, an ‘empty vector' control (all shown in Figure 6A) or in the absence of protein. The experiment was performed as in Figure 6B, only a buffer containing 3 mM manganese instead of 100 μM magnesium was used. The final hDIS3 protein concentration was 0.25 μM. (B) hDIS3L^{RNB MUT} is catalytically inert. 5′-labelled ss17-A₃₀ (left) or circularized ss17-A₁₄ (right) substrates were incubated with hDIS3L^{RNB MUT}, hDIS3L^DM or in the absence of protein (shown in Figure 6A). Where indicated, an ‘empty vector' control and hDIS3^{RNB MUT} protein diluted to the same concentration as hDIS3L versions (0.04 μM) were also used. The experiment was performed as in (A).

Figure 8

Differential compartmentalization of human exosome isoforms. Overview of the localization of catalytic exosome subunits. The ubiquitously present catalytically inert exosome core associates in the nucleus with the processive 3′ → 5′ exoRNase and endoRNase hDIS3, and with the distributive 3′ → 5′ exoRNase hRRP6. In the nucleolus, the core exosome primarily binds hRRP6. Finally, in the cytoplasm, the core associates with the cytoplasmic-restricted processive 3′ → 5′ exoRNase hDIS3L, which is devoid of endoRNase activity. Both hRRP6 and hDIS3 are also present in the cytoplasm, albeit in lower amounts than in the nucleus/nucleolus. It remains to be determined whether free core particles and catalytic subunits exist.