Interaction between PATL1 and EDC4 requires the presence of the C-terminus of PATL1 and the N-terminus of EDC4.

After transfection of a plasmid encoding GFP-PATL1 into HEp-2 cells, endogenous EDC4 (panel a) and GFP-PATL1 (panels b, c) localized to cytoplasmic dots. Co-expression of NLS-EDC4 and GFP-PATL1 in HEp-2 cells resulted in localization of NLS-EDC4 (panel d) in nuclear dots, while GFP-PATL1 remained in cytoplasmic dots (panels e, f). Expression of a plasmid encoding NLS-EDC4 (panel g) together with a plasmid encoding Myc fused to PATL1 amino acids 398–770 (panels h, i) resulted in the two proteins co-localizing in nuclear dots in 97 +/-3% of cells that expressed both proteins. When cells were transfected with GFP-NLS-EDC4(630–1437) (panel j) and Myc-PATL1(398–770) (panels k, l), GFP-NLS-EDC4(630–1437) localized to nuclear dots while Myc-PATL1(398–770) remained in cytoplasmic dots in all cells that expressed both proteins. Note that the anti-Myc antiserum produces faint diffuse nuclear staining. EDC4 in panels a, d, and g was detected using human serum containing anti-EDC4 antibodies. GFP-PATL1 in panels b and e, and GFP-NLS-EDC4(630–1437) in panel j were detected using mouse monoclonal anti-GFP antibodies. Myc-PATL1 in panels h and k was detected using rabbit anti-Myc antiserum. Merge of panels a and b, d and e, g and h, and j and k is shown in c, f, i and l respectively. DAPI staining (blue) indicates location of nuclei in c, f, i and I. White arrows in j-l indicate a representative cell that contains NLS-EDC4(630–1437) in nuclear dots, while Myc-PATL1(398–770) localized to cytoplasmic dots.</p

Effect of an exogenous nuclear localization sequence (NLS) on the cellular location of P-body components.

Expression of NLS-EDC4 (panels a, b) and GFP-NLS-EDC4 (panels c, d) in HEp-2 cells resulted in localization of the protein to nuclear dots. In contrast, diffuse nuclear staining was seen after transfection of plasmids encoding NLS fused to DCP1a (panels e, f), LSm14a (panels g, h) or DDX6 amino acids (289–483) (panels k and l). Addition of a NLS to the N-terminus of XRN1 amino acids 1232–1706 had no effect on the distribution of the protein fragment, which localized to cytoplasmic dots (panels I, j). Human serum was used to detect EDC4 in panel a. Mouse anti-GFP antibody detected GFP-NLS-EDC4 in panel c. Rabbit antiserum was used to detect NLS-DCP1a (panel e) or NLS-LSm14a (panel g). Rabbit anti-monomeric cherry (mCh) antiserum was used to detect mCh-NLS-XRN1(1232–1706) and mCh-NLS-DDX6(289–483) in i and k, respectively. DAPI staining in b, d, f, h, j and l indicates the location of nuclei in the preceding panels. (TIF)</p

Model of the mammalian mRNA P-body.

Interactions between EDC4 and LSm14a, PATL1, XRN1, MARF1, NBDY and DDX6 (via PATL1) require the N-terminal, WD40-containing domain in EDC4. The C-terminus of EDC4 is sufficient to mediate interaction between EDC4 and DCP2, DCP1a, CCHCR1 and EDC3 (via DCP1a). RNA binding proteins DDX6, LSm14a, PATL1, and MARF1 may be present on the “outer surface” of the P-body, while the decapping protein (DCP2) and enhancers of decapping may be “buried”, and perhaps inactive, inside P-bodies. Phosphorylation of NBDY or depletion of LSm14a or DDX6 results in disruption of P-bodies.</p

When expressed in HEp-2 cells, GFP-NLS-EDC4 (panel a) localized to Cajal bodies (panels b, c).

Co-expression of NLS-EDC4 (panel d) and mCh-XRN1(1232–1706) (panel e) resulted in localization of NLS-EDC4 to nuclear dots, while mCh-XRN1(1232–1706) localized to cytoplasmic dots. Expression of GFP-NLS-EDC4(630–1437) (panel g) and CCHCR1-Myc (panel h) in HEp-2 cells resulted in localization of both proteins to nuclear dots. Co-expression of NLS-EDC4 and mCh-NLS-DDX6(289–483) in HEp-2 cells resulted in localization of NLS-EDC4 to nuclear dots (panel j), while mCh-NLS-DDX6(289–483) was distributed diffusely throughout the nucleus (panel k). Mouse monoclonal anti-GFP antibody was used to detect GFP-NLS-EDC4 (panel a) and GFP-NLS-EDC4(630–1437) (panel g). Human serum containing anti-Cajal antibodies was used in panel b. Human serum containing anti-EDC4 antibodies was used to detect NLS-EDC4 in (panels d and j). Rabbit anti-mCh antiserum was used to detect mCh-XRN1(1232–1706) and mCh-NLS-DDC6(289–483) (panels e and k). Rabbit anti-Myc antiserum was used to detect CCHCR1-Myc in panel h. Merge of panels a and b, d and e, g and h, j and k is shown in c, f, i, and l, respectively. DAPI staining in f and i indicate the location of nuclei. (TIF)</p

Endogenous LSm14a and DDX6 did not co-localize with NLS-EDC4 in nuclear dots.

After expression of a plasmid encoding NLS-EDC4 in HEp-2 cells, human serum containing anti-EDC4 antibodies detected EDC4 in both nuclear and cytoplasmic dots (panels a and d). Endogenous LSm14a (panel b) and DDX6 (panel e) were only detected in cytoplasmic dots. Rabbit anti-LSm14a and anti-DDX6 antisera were used to detect the corresponding proteins. Merge of panels a and b, and c and e, is shown in c and f, respectively. DAPI staining in c and f indicate the location of nuclei in the preceding panels. (TIF)</p

EDC4(630–1437) can recruit DCP1a, EDC3 or CCHCR1 to cytoplasmic dots despite depletion of LSm14a and loss of endogenous P-bodes.

After treatment with scrambled siRNA, LSm14a (panel a), EDC4(630–1437) (panel b) and GFP-DCP1a (panel c) all localized in cytoplasmic dots. siRNA directed against LSm14a resulting in depletion of LSM14a from HEp-2 cells (panel d), but EDC4(630–1437) (panel e) and GFP-DCP1a (panel f) localized to cytoplasmic dots. Depletion of LSm14a (panels g, j) did not affect localization of EDC4(630–1437) (panels h, k) and EDC3 (panel i) or CCHCR1 (panel l) to cytoplasmic dots. Rabbit antiserum was used to detect LSm14a in panel a and to confirm the absence of LSm14a and endogenous P-bodies in d, g, and j. Human serum reacted with EDC4(630–1437) in b, e, h and k. Mouse anti-GFP antibody detected GFP-DCP1a in c and f, GFP-EDC3 in i, and CCHCR1-GFP in l.</p

NLS-EDC4 localizes to Cajal bodies, and adjacent to PML-nuclear bodies.

A plasmid encoding green fluorescent protein (GFP) fused to the nuclear localization sequence (NLS) of SV40 T antigen and full length EDC4 was transfected into HEp-2 cells. GFP-NLS-EDC4 (panel a) localized adjacent to PML-nuclear bodies (panels b, c). The GFP-NLS-EDC4 fusion protein was detected using mouse monoclonal anti-GFP antibodies and PML nuclear bodies were identified using rabbit anti-Sp100 antiserum. Transfection of GFP-NLS-EDC4 and subsequent staining with mouse anti-GFP antibodies and rabbit anti-p80 coilin antibodies revealed that NLS-EDC4 (panel d) localized to Cajal bodies (panels e, f). Transfection of HEp-2 cells with a plasmid encoding GFP-NLS-EDC4(630–1437) (panel g) and subsequent staining with mouse anti-GFP antibodies and human serum containing anti-p80 coilin antibodies (panels h, i), revealed that in the absence of the N-terminus, EDC4 was no longer able to localize to Cajal bodies. GFP-NLS-EDC4(630–1437) (panel j) retained the ability to localize adjacent to PML nuclear bodies (panels k, l). Merge of panels a and b, d and e, g and h, and j and k is shown in panels c, f, i, and l respectively. DAPI staining (blue) indicates the location of nuclei in c, f, i and l.</p

Reagents and resources.

Messenger RNA processing bodies (P-bodies) are cytoplasmic membrane-free organelles that contain proteins involved in mRNA silencing, storage and decay. The mechanism by which P-body components interact and the factors that regulate the stability of these structures are incompletely understood. In this study, we used a fluorescence-based, two-hybrid assay to investigate interactions between P-body components that occur inside the cell. LSm14a, PATL1, XRN1, and NBDY were found to interact with the N-terminal, WD40-domain-containing portion of EDC4. The N-terminus of full-length PATL1 was required to mediate the interaction between EDC4 and DDX6. The C-terminal, alpha helix-domain- containing portion of EDC4 was sufficient to mediate interaction with DCP1a and CCHCR1. In the absence of endogenous P-bodies, caused by depletion of LSm14a or DDX6, expression of the portion of EDC4 that lacked the N-terminus retained the ability to form cytoplasmic dots that were indistinguishable from P-bodies at the level of UV light microscopy. Despite the absence of endogenous P-bodies, this portion of EDC4 was able to recruit DCP1a, CCHCR1 and EDC3 to cytoplasmic dots. The results of this study permit the development of a new model of P-body formation and suggest that the N-terminus of EDC4 regulates the stability of these structures.</div

The N-terminus of EDC4 is not required for interaction with DCP1a.

Co-expression of NLS-EDC4 (panel a) and NLS-DCP1a (panel b) resulted in co-localization of the proteins to nuclear dots (panel c) in all cells that had both proteins in the nucleus. White arrows in a-c indicate a representative cell containing both NLS-EDC4 and NLS-DCP1a in nuclear dots. GFP-NLS-EDC4(630–1437) (panel d) was also able to recruit NLS-DCP1a (panels e, f) to nuclear dots in all cells that expressed both proteins. The C-terminus of EDC4 is sufficient to mediate interaction with CCHCR1. Co-expression of NLS-EDC4 (panel g) and CCHCR1-Myc (panel h) resulted in co-localization of the proteins in nuclear dots (panel i) in all cells that expressed both proteins. The C-terminus of EDC4 (GFP-NLS-EDC4(935–1437)) (panel j) was sufficient to recruit CCHCR1-Myc (panels k, l) to nuclear dots. CCHCR1-Myc co-localized with GFP-NLS-EDC4(935–1437) in 96+/-4% of cells expressing both proteins. White arrows indicate the location of representative cells that expressed NLS-EDC4 and CCHCR1-Myc (g-i) and NLS-EDC4(935–1437) and CCHCR1-Myc (j-l). Human serum was used to detect NLS-EDC4 in a and g. Rabbit antiserum was used to detect NLS-DCP1a in b and e. Rabbit anti-Myc antiserum was used to detect CCHCR1-Myc in h and k. Mouse monoclonal anti-GFP antibody detected GFP-NLS-EDC4(630–1437) and GFP-NLS-EDC4(935–1437) in d and j respectively. Merge of panels a and b, d and e, g and h, and j and k is shown in c, f, i and l respectively. DAPI staining (blue) indicates the location of nuclei in c, f, i and l.</p

After depletion of LSm14a and loss of endogenous P-bodies, GFP-EDC4(630–1437), but not GFP-EDC4, localizes to cytoplasmic dots.

siRNA directed against LSm14a was transfected into HEp-2 cells, followed 24 hours later by transfection of GFP-EDC4 or GFP-EDC4(630–1437). LSm14a was not detected in siLSm14a-treated cells (panel a). GFP-EDC4 (panels b, c) was unable to localize to P-bodies and was distributed throughout the cytoplasm of transfected cells. In cells depleted of LSm14a (panel d) and expressing GFP-EDC4(630–1437) (panels e, f), GFP-EDC4(630–1437) was detected in cytoplasmic dots that were indistinguishable from P-bodies at the level of UV light microscopy. After disruption of endogenous P-bodies, neither full-length EDC4 nor EDC4(630–1437) was able to recruit DDX6 to cytoplasmic dots. After depletion of LSm14a using siRNA, GFP-EDC4 (panel g) did not form cytoplasmic dots and was unable to recruit endogenous DDX6 (panel h) to cytoplasmic dots. After depletion of LSm14a, GFP-ED4(630–1437) (panel j) was able to form cytoplasmic dots, but was unable to recruit endogenous DDX6 (panels k, l) to these structures. Rabbit antiserum was used to confirm the absence of LSm14a in a and d. Mouse anti-GFP antibodies detected GFP-EDC4 (b, g) and GFP-EDC4(630–1437) (e and j). Rabbit anti-DDX6 antiserum was used to detect endogenous DDX6 in h and k. Merge of panels a and b, d and e, g and h, and j and k is shown in c, f, i, and l, respectively. DAPI staining (blue) in c, f, i and l indicate the location of nuclei.</p