Three Novel Components of the Human Exosome

Abstract

The yeast exosome is a complex of 3 3 5 exoribonucleases. Sequence analysis identified putative human homologues for exosome components, although several were found only as expressed sequence tags. Here we report the cloning of full-length cDNAs, which encode putative human homologues of the Rrp40p, Rrp41p, and Rrp46p components of the exosome. Recombinant proteins were expressed and used to raise rabbit antisera. In Western blotting experiments, these decorated HeLa cell proteins of the predicted sizes. All three human proteins were enriched in the HeLa cells nucleus and nucleolus, but were also clearly detected in the cytoplasm. Size exclusion chromatography revealed that hRrp40p, hRrp41p, and hRrp46p were present in a large complex. This cofractionated with the human homologues of other exosome components, hRrp4p and PM/ Scl-100. Anti-PM/Scl-positive patient sera coimmunoprecipitated hRrp40p, hRrp41p, and hRrp46p demonstrating their physical association. The immunoprecipitated complex exhibited 3 3 5 exoribonuclease activity in vitro. hRrp41p was expressed in yeast and shown to suppress the lethality of genetic depletion of yeast Rrp41p. We conclude that hRrp40p, hRrp41p, and hRrp46p represent novel components of the human exosome complex.In both bacteria and eukaryotes, the processing and degradation of many RNA species involves multiprotein complexes (reviewed in Refs. 1-4). The Escherichia coli degradosome includes the endoribonuclease E (RNase E), the 3Ј 3 5Ј exonuclease polynucleotide phosphorylase, the DEAD box RNA helicase RhlB, and several additional proteins whose role is unclear (5-7). Related complexes are implicated in RNA processing in chloroplasts and mitochondria (8 -10). The yeast exosome contains at least 11 components, which are known or predicted to be 3Ј 3 5Ј exoribonucleases (11,12). Ten of these (Rrp4p, Rrp40 -46p, Mtr3p, and Csl4p) have been demonstrated to be encoded by essential genes. These 10 components were found in both cytoplasmic and nuclear complexes, whereas the nonessential RRP6 gene product was detected only in the nucleus (11, 13).The 3Ј processing of many RNAs is affected by the absence or mutation of exosome components. The nuclear exosome is implicated in the processing of ribosomal RNA (rRNA), spliceosomal small nuclear RNAs, and small nucleolar RNAs, as well as the degradation of pre-rRNA spacers and unspliced pre-mRNAs (12-22). The cytoplasmic exosome complex is involved in the 3Ј 3 5Ј pathway of mRNA degradation (22). The activity of the exosome complex may be regulated by cofactors including, for example, the putative ATP-dependent DEVH box RNA helicases Dob1p and Ski2p (23,24).Human cells also contain a multiprotein complex that is related to the yeast exosome (11). This complex, initially designated as the polymyositis/scleroderma (PM/Scl) 1 overlap syndrome particle and herein referred to as the human exosome, was reported to contain 11 (25) to 16 (26) subunits ranging from 20 to 110 kDa. Two proteins of this complex were identified as autoantigens, which are tar...

A Genome-wide Short Hairpin RNA Screening of Jurkat T-cells for Human Proteins Contributing to Productive HIV-1 Replication

“…STXBP2 is an interactor of SYNTAXIN 3, which acts on the membrane function and processes of the cell (23); however, it is unclear how knockdown of STXBP2 (directly or indirectly) affects the amount of reverse transcribed HIV-1 proviral DNA. EXOSC5 has been reported to be involved in RNA processing (24). Intriguingly, detailed proteomic data show that this protein interacts with nucleoporin (NUP210), a component of the nuclear pore complex (25), and a novel protein KIAA1217, which engages the TSG101-ESCRT and trans-Golgi pathways (supplemental Fig.…”

Post‐transcriptional regulation of gene expression by degradation of messenger RNAs

“…This complex, afterwards referred to as the human exosome, was reported to contain 11-16 subunits ranging from 20 to 110 kDa (Reimer et al, 1986;Gelpi et al, 1990). Size-exclusion chromatography of the human exosome complex in a HeLa cell extract indicated that the cytoplasmic complex has a molecular mass of about 700 kDa (Brouwer et al, 2001); the size previously estimated by glycerol density gradient centrifugation was 300-400 kDa (Mitchell et al, 1997).…”

ZAP-mediated mRNA degradation

“…RNA helicase KIAA0052 has been reported to associate with the exosome but its role in exosome-mediated RNA degradation is not fully elucidated yet. 13,14 In ARE mediated mRNA decay, RNA helicase RHAU is temporarily recruited to the exosome to facilitate degradation of ARE-containing mRNAs. 14 Recently, the RNA helicase p72 was identified as a factor required for optimal function of ZAP.…”