Autoantibody to hLSm4 and the heptameric LSm complex in anti‐Sm sera

Eystathioy, Theophany; Peebles, Carol; Hamel, John C.; Vaughn, John; Chan, Edward K. L.

doi:10.1002/art.10220

Cited by 20 publications

(8 citation statements)

References 26 publications

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“…6d, lane 3 Consistent with its functions in splicing and rRNA processing, the LSm2-8 complex has been found to localize in the nucleus (34), and in the nucleolus (35). LSm1-7 accumulates in particular cytoplasmic features called foci (28) or GW bodies (36,37). To test whether our recombinant LSm complexes show corresponding subcellular distributions, we injected fluorescently labeled LSm1-7 and LSm2-8 into rat fibroblasts.…”

Section: Resultsmentioning

confidence: 89%

Reconstitution of Two Recombinant LSm Protein Complexes Reveals Aspects of Their Architecture, Assembly, and Function

Zarić

Chami

Rémigy

et al. 2005

Journal of Biological Chemistry

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Sm and Sm-like (LSm) proteins form complexes engaging in various RNA-processing events. Composition and architecture of the complexes determine their intracellular distribution, RNA targets, and function. We have reconstituted the human LSm1-7 and LSm2-8 complexes from their constituent components in vitro. Based on the assembly pathway of the canonical Sm core domain, we used heterodimeric and heterotrimeric sub-complexes to assemble LSm1-7 and LSm2-8. Isolated sub-complexes form ring-like higher order structures. LSm1-7 is assembled and stable in the absence of RNA. LSm1-7 forms ring-like structures very similar to LSm2-8 at the EM level. Our in vitro reconstitution results illustrate likely features of the LSm complex assembly pathway. We prove the complexes to be functional both in an RNA bandshift and an in vivo cellular transport assay.The Sm and Sm-like (LSm) 1 proteins are a widespread protein family with members in all kingdoms of life. Phylogenetic distribution suggests Sm proteins were already present in the last universal common ancestor of all present-day life forms and that the family underwent an explosive diversification with the advent of eukaryotes (1). Archaebacteria harbor one or two SM/LSM genes each. Escherichia coli Hfq and its orthologues in other Gram-negative bacteria are so far the only known eubacterial LSm proteins (2, 3). In contrast, eukaryotic genomes appear to contain 24 or more Sm/LSm genes (1, 4). Thought to have originally arisen as chaperones mediating RNA-RNA interactions (5), Sm/LSm proteins have diversified through evolution and adopted new functionalities. LSm protein function in archaea is unknown. A structural and biochemical study on Archaeoglobus fulgidus LSm proteins showed that they bind to RNase P RNA in vivo and in vitro (6), a feature that has also been observed for several yeast LSm proteins (7). E. coli Hfq is a pleiotropic regulator of RNA metabolism (8).The originally identified canonical Sm proteins engage in pre-mRNA splicing (9). Sm/LSm family members have been shown to participate in mRNA decapping and degradation (10, 11), histone pre-mRNA processing (12-14), telomere synthesis (15), rRNA maturation (16, 17), small nucleolar ribonucleoprotein assembly (18), pre-tRNA processing (19), and trans-splicing (20,21).Sm/LSm proteins are characterized by a bipartite sequence motif of about 80 amino acids long situated in most members at the N terminus. Recently, divergent family members with additional domains have been identified (1, 4). The conserved motif translates into a fold common to all Sm/LSm proteins. This Sm fold mediates specific Sm-Sm interaction through a generic interface, which the various Sm protein family members use to build up homomeric (in prokaryotes) or heteromeric (in eukaryotes) ring-shaped complexes. These represent the functional form of all Sm/LSm proteins. The common fold, generic interface, and ring-like morphology of Sm/LSm complexes provide a rationale for the observed large variety of RNA targets bound, the diverse complex composi...

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Section: Resultsmentioning

confidence: 89%

Reconstitution of Two Recombinant LSm Protein Complexes Reveals Aspects of Their Architecture, Assembly, and Function

Zarić

Chami

Rémigy

et al. 2005

Journal of Biological Chemistry

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“…Rabbit anti-Ago2 was a gift from Dr. Tom Hobman (University of Alberta, Edmonton, AB, Canada), and rabbit anti-Dcp1a was obtained from Dr. Jens Lykke-Andersen (University of Colorado). Rabbit polyclonal anti-LSm4 was produced as described previously (Eystathioy et al, 2002b). Mouse monoclonal anti-lamin A/C 636, anti-TIAR, and anti-tubulin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA), BD Biosciences (San Jose, CA), and SigmaAldrich (St. Louis, MO), respectively.…”

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confidence: 99%

Small Interfering RNA-mediated Silencing Induces Target-dependent Assembly of GW/P Bodies

Lian¹,

Fritzler

Katz³

et al. 2007

MBoC

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Gene silencing using small interfering RNA (siRNA) is a valuable laboratory tool and a promising approach to therapeutics for a variety of human diseases. Recently, RNA interference (RNAi) has been linked to cytoplasmic GW bodies (GWB). However, the correlation between RNAi and the formation of GWB, also known as mammalian processing bodies, remains unclear. In this report, we show that transfection of functional siRNA induced larger and greater numbers of GWB. This siRNA-induced increase of GWB depended on the endogenous expression of the target mRNA. Knockdown of GW182 or Ago2 demonstrated that the siRNA-induced increase of GWB required these two proteins and correlated with RNAi. Furthermore, knockdown of rck/p54 or LSm1 did not prevent the reassembly of GWB that were induced by and correlated with siRNA-mediated RNA silencing. We propose that RNAi is a key regulatory mechanism for the assembly of GWB, and in some cases, GWB may serve as markers for RNAi in mammalian cells. INTRODUCTIONGW bodies (GWB), also known as mammalian processing bodies (P bodies), are cytoplasmic foci that contain multiple decay factors and that are involved in the 5Ј33Ј mRNA degradation pathway. GWB are named from the marker protein GW182, which contains multiple glycine (G) and tryptophan (W) repeats and a classic RNA binding domain at the carboxyl terminus (Eystathioy et al., 2002a). The mRNA decay factors/complexes found in GWB include the deadenylase Ccr4, the decapping complex Dcp1a/1b/Dcp2, the LSm1-7 complex, Ge-1 (also known as Hedls), rck/p54, and exonuclease Xrn1 (Bashkirov et al., 1997;van Dijk et al., 2002;Ingelfinger et al., 2002;Lykke-Andersen, 2002;Eystathioy et al., 2003;Cougot et al., 2004;Andrei et al., 2005;Yu et al., 2005;Fenger-Gron et al., 2005). GWB are physically juxtaposed to and transiently interact with stress granules (SG). SG process cytoplasmic aggregates of stalled translational preinitiation complexes that accumulate during stress responses and that share certain components with GWB (Kedersha et al., 2005).In addition to mRNA decay, a crucial role of GWB and their components in RNA interference (RNAi) was recently uncovered (Anderson and Kedersha, 2006;Eulalio et al., 2007a;Jakymiw et al., 2007). RNAi is a posttranscriptional gene silencing mechanism that uses specific doublestranded RNA to silence genes in a sequence-specific manner Mello and Conte, 2004). In brief, the double-stranded RNA is processed by Dicer into small interfering RNA (siRNA) or microRNA (miRNA). The 21-to 26-nucleotide siRNA and miRNA are then incorporated in the effector complex, RNA-induced silencing complex (RISC), which either cleaves or inhibits translation of the target mRNA. In 2005, two key components of RISC, Argonaute2 (Ago2) and siRNA/miRNA, were found to be enriched in GWB (Sen and Blau, 2005;Pillai et al., 2005;Jakymiw et al., 2005;Liu et al., 2005b;Pauley et al., 2006). miRNA-targeted mRNA also localizes to GWB in a miRNAdependent manner (Liu et al., 2005b). These observations provide the first evidence that RNAi is ...

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“…plasmic foci (Tharun and Parker 2001;Eystathioy et al 2002b;van Dijk et al 2002). Two proteins of the heptameric hLSm complex, hLSm1 and hLSm4 were among the proteins localized to such foci (Ingelfinger et al 2002).…”

Section: Resultsmentioning

confidence: 99%

The GW182 protein colocalizes with mRNA degradation associated proteins hDcp1 and hLSm4 in cytoplasmic GW bodies: FIGURE 1.

Eystathioy¹,

Jakymiw²,

Chan³

et al. 2003

RNA

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A novel cytoplasmic compartment referred to as GW bodies (GWBs) was initially identified using antibodies specific to a 182-kD protein termed GW182. GW182 was characterized by multiple glycine(G)-tryptophan(W) repeats and an RNA recognition motif (RRM) that bound a subset of HeLa cell messenger RNAs (mRNAs). The function of GWBs was not known; however, more recent evidence suggested similarities between GWBs and cytoplasmic structures that contain hLSm proteins and hDcp1, the human homolog to a yeast decapping enzyme subunit. In this study, we used antibodies to hLSm4 and hDcp1 to show that both of these markers of an mRNA degradation pathway colocalize to the same structures as GW182. Our studies demonstrate that GW182, hLSm4, and hDcp1 are found in the same cytoplasmic structures and suggest that GW182 is involved in the same mRNA processing pathway as hLSm4 and hDcp1.

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Autoantibody to hLSm4 and the heptameric LSm complex in anti‐Sm sera

Cited by 20 publications

References 26 publications

Reconstitution of Two Recombinant LSm Protein Complexes Reveals Aspects of Their Architecture, Assembly, and Function

Reconstitution of Two Recombinant LSm Protein Complexes Reveals Aspects of Their Architecture, Assembly, and Function

Small Interfering RNA-mediated Silencing Induces Target-dependent Assembly of GW/P Bodies

The GW182 protein colocalizes with mRNA degradation associated proteins hDcp1 and hLSm4 in cytoplasmic GW bodies: FIGURE 1.

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