CERKL, a retinal disease gene,encodes an mRNA-biding protein that localizes in compact and unstranslated mRNPs associated to microtubules.

The function of CERKL (CERamide Kinase Like), a causative gene of retinitis pigmentosa and cone-rod dystrophy, still awaits characterization. To approach its cellular role we have investigated the subcellular localization and interaction partners of the full length CERKL isoform, CERKLa of 532 amino acids, in different cell lines, including a photoreceptor-derived cell line. We demonstrate that CERKLa is a main component of compact and untranslated mRNPs and that associates with other RNP complexes such as stress granules, P-bodies and polysomes. CERKLa is a protein that binds through its N-terminus to mRNAs and interacts with other mRNA-binding proteins like eIF3B, PABP, HSP70 and RPS3. Except for eIF3B, these interactions depend on the integrity of mRNAs but not of ribosomes. Interestingly, the C125W CERKLa pathological mutant does not interact with eIF3B and is absent from these complexes. Compact mRNPs containing CERKLa also associate with microtubules and are found in neurites of neural differentiated cells. These localizations had not been reported previously for any member of the retinal disorders gene family and should be considered when investigating the pathogenic mechanisms and therapeutical approaches in these diseases

Eukaryotic Initiation Factor 5A2 localizes to actively translating ribosomes to promote cancer cell protrusions and invasive capacity

[EN] Background Eukaryotic Initiation Factor 5A (eIF-5A), an essential translation factor, is post-translationally activated by the polyamine spermidine. Two human genes encode eIF-5A, being eIF5-A1 constitutively expressed whereas eIF5-A2 is frequently found overexpressed in human tumours. The contribution of both isoforms with regard to cellular proliferation and invasion in non-small cell lung cancer remains to be characterized. Methods We have evaluated the use of eIF-5A2 gene as prognosis marker in lung adenocarcinoma (LUAD) patients and validated in immunocompromised mice. We have used cell migration and cell proliferation assays in LUAD lines after silencing each eIF-5A isoform to monitor their contribution to both phenotypes. Cytoskeleton alterations were analysed in the same cells by rhodamine-phalloidin staining and fluorescence microscopy. Polysome profiles were used to monitor the effect of eIF-5A2 overexpression on translation. Western blotting was used to study the levels of eIF-5A2 client proteins involved in migration upon TGFB1 stimulation. Finally, we have co-localized eIF-5A2 with puromycin to visualize the subcellular pattern of actively translating ribosomes. Results We describe the differential functions of both eIF-5A isoforms, to show that eIF5-A2 properties on cell proliferation and migration are coincident with its features as a poor prognosis marker. Silencing of eIF-5A2 leads to more dramatic consequences of cellular proliferation and migration compared to eIF-5A1. Overexpression of eIF5A2 leads to enhanced global translation. We also show that TGF ss signalling enhances the expression and activity of eIF-5A2 which promotes the translation of polyproline rich proteins involved in cytoskeleton and motility features as it is the case of Fibronectin, SNAI1, Ezrin and FHOD1. With the use of puromycin labelling we have co-localized active ribosomes with eIF-5A2 not only in cytosol but also in areas of cellular protrusion. We have shown the bulk invasive capacity of cells overexpressing eIF-5A2 in mice. Conclusions We propose the existence of a coordinated temporal and positional interaction between TFGB and eIF-5A2 pathways to promote cell migration in NSCLC. We suggest that the co-localization of actively translating ribosomes with hypusinated eIF-5A2 facilitates the translation of key proteins not only in the cytosol but also in areas of cellular protrusion.This work was supported by: Fondo de Investigacion Sanitaria, ISCIII, grant number PI20-194, co-funded by ERDF/ESF, "Investing in your future". Ministerio de Educacion, Cultura y Deporte grant FPU13/02755 for JMPS. Asociacion Espanola contra el Cancer, AECC predoctoral grant for AMF. Part of the equipment employed in this work has been funded by Generalitat Valenciana and co-financed with ERDF funds (OP ERDF of Comunitat Valenciana 2014-2020). This article is based upon work from COST Action CA20113 ProteoCure, supported by COST (European Cooperation in Science and Technology).Martínez-Férriz, A.; Gandía, C.; Pardo-Sánchez, JM.; Fathinajafabadi, A.; Ferrando Monleón, AR.; Farras, R. (2023). Eukaryotic Initiation Factor 5A2 localizes to actively translating ribosomes to promote cancer cell protrusions and invasive capacity. Cell Communication and Signaling. 21(1). https://doi.org/10.1186/s12964-023-01076-621

Aproximacion funcional a CERKL, un gen causante de la retinosis pigmentaria, mediante el estudio de la localización intracelular de la proteína y la identificación de sus partners proteicos y no proteicos

La proteína CERKL se encuentra localizada tanto en el núcleo como en el citoplasma. En el citoplasma, CERKL se distribuye de manera difusa y también formando unos agregados que en su mayor parte se distribuyen en la periferia nuclear. Esos agregados colocalizan con marcadores de gránulos de estrés y su cantidad aumenta sometiendo las células a estrés (arsenito de sodio o choque térmico) y disminuye tras un tratamiento con cicloheximida (un inhibidor de la síntesis de proteínas que desensambla los gránulos de estrés). La entrada y salida de CERKL del núcleo es necesaria para su localización en los gránulos de estrés y, por eso, el mutante patológico C125W que está ausente de los núcleos no colocaliza con los marcadores de gránulos de estrés. CERKL se acumula en el núcleo y su presencia en los gránulos de estrés disminuye considerablemente cuando se tratan las células con leptomicina B (un inhibidor de la salida de proteínas del núcleo) o con actinomicina D y α-amanitina (inhibidores de la transcripción). CERKL colocaliza también con otros componentes celulares conteniendo ribonucleoproteínas, como son los cuerpos P, los polisomas y unas partículas ribonucleoproteicas compactas. La asociación de CERKL en los polisomas se pierde cuando se tratan con EDTA y RNasa A que los desensamblan, mientras que en las partículas ribonucleoproteicas compactas eso solo ocurre cuando los tratamientos anteriores se realizan a concentraciones salinas elevadas que reducen su compactación. CERKL interacciona con: i) proteínas de la maquinaria de traducción de los RNAs mensajeros, como son eIF3B, PABP o RPS3, ii) chaperonas que intervienen en el plegamiento de las proteínas recién sintetizadas, como son HSP70 o HSP90, iii) las tubulinas α y β de los microtúbulos, y iv) proteínas de la membrana plasmática, como son la filagrina, la desmoplaquina y la desmogleína. Además, CERKL interacciona con proteínas que intervienen en el metabolismo del DNA o del RNA, como la nucleofosmina, en el transporte al núcleo, como la importina 4, y en la unión a lípidos, como la apolipoproteína D. La interacción de CERKL con PABP, RPS3 y HSP70, pero no con eIF3B, se pierde tras un tratamiento con RNasa A en presencia de concentraciones salinas elevadas. Estos resultados son consistentes con que la mayoría de esas interacciones ocurren en partículas ribonucleoproteicas compactas y que la interacción de CERKL con PABP, RPS3 y HSP70 depende de la integridad de los RNAs mensajeros, mientras que la interacción con eIF3B podría ocurrir directamente. El mutante patológico C125W de CERKL es incapaz de interaccionar con la proteína eIF3B y sus interacciones con PABP, HSP70 y RPS3 no se pierden tras un tratamiento con RNasa A. Esto sugiere que este mutante presenta anormalidades en la formación de las partículas ribonucleoproteicas compactas, probablemente debido a su incapacidad de unirse eficazmente a los RNAs mensajeros. CERKL interacciona con componentes del complejo 48S de preinicio de la traducción, tales como las proteínas eIF3B, eIF3G y eIF3I, con proteínas ribosomales de la subunidad 40S, como las RPS3, RPS5 y RPSA y con factores no proteicos, como el residuo 7-metil-guanosina del extremo 5´ del RNA mensajero.Within the last two and a half decades, technological advances have led to a progress in gene identification of retinal diseases. However, the function of a high number of the over hundred retinal diseases and fifty RP genes known remains to be characterized. Functional studies have been hampered mainly by the lack of suitable animal and cellular models, the intracellular complexity and specific metabolic demands of the retinal cells and the extensive genetic, allelic and clinical heterogeneity associated with the majority of retinal degenerative disorders. Within this context, CERKL, an autosomal recessive RP and CRD -causing gene, stands among the genes whose function awaits characterization. In fact, repeated attempts by several groups to assign a ceramide kinase function to CERKL have proved fruitless. Therefore and to gain some insight into CERKL function we investigated here in detail the subcellular localization and the interaction partners of the CERKL full length protein isoform (CERKLa). The protein was localized both in the nucleus and in the cytoplasm of various cell types, in agreement with other studies. Although the CERKL distribution appeared to be mainly homogeneous, some aggregates became apparent in the cytoplasm of transiently transfected cells. A systematic analysis in cultured cells using different markers of various cell compartments revealed that CERKL aggregates colocalized with SGs, which are cytoplasmic complexes of mRNAs and mRNA-binding proteins such as those that regulate mRNA translation and stability. These granules are induced by stress conditions and provide the cells with a mechanism to stop protein synthesis and promote a quick recovery of proteostasis when the stress disappears. Targeting to SGs under stress conditions would agree with CERKL protective role from apoptosis induced by oxidative stress. In addition to SGs, CERKL was also found associated with P-bodies, which are involved in mRNA degradation and are in dynamic equilibrium with SGs. Also in support to a CERKL contribution to stress response and protection of photoreceptors is the punctuated CERKL labeling noticed after a light stress in the outer nuclear layer of rat photoreceptors. The CERKL-C125W mutant, which does not enter the nucleus, is not found in SGs. In fact, the localization of wild type CERKL to SGs seems to depend on nuclear import/export of the protein and on mRNA transcription. Therefore, we reasoned that CERKL has a role in the nucleus before it associates with SGs, probably related to RNA transport. In fact, CERKL subcellular localization studies have shown strong accumulation in the nucleoli. Whether this is related to particular protein isoforms or depends on the cellular state deserves further study. Actually, there are other genes mutated in RP that are involved in RNA metabolism at the nucleus, such as the spliceosome components PRPF31, PRPF8 and PRPF3, or the RNA splicing factor RP9. Recently, in age-related macular degeneration, SFRS10 has been reported to regulate alternative splicing of stress response genes under hypoxic conditions. In the cytosol, apart from the localization of CERKL in aggregates, the protein was mostly found diffusely distributed. This was particularly evident in HeLa cells stably expressing this protein. Since SGs are in dynamic equilibrium with polysomes, we analyzed whether this diffuse localization of CERKL could correspond to its binding to polysomes. Using sucrose gradients, CERKL localized to the polysomal pellet and to a greater extent in the soluble fractions that contained postpolysomal mRNPs. Contrary to polysomes, the association of CERKL to these fractions was not sensitive to EDTA or puromycin and was only sensitive to RNase A at high salt concentrations. Therefore, it appears that CERKL in these fractions is associated with quite compact mRNPs. The association of CERKL to mRNPs can occur by protein-RNA and protein-protein interactions. Our data show that CERKL directly interacts with mRNA through its N-terminal region and that this link involves the 5′cap structure of mRNA. Since in the proteomic analysis we did not observe any interaction between CERKL and the two mammalian proteins known to bind directly to the 5′cap structure, elF4E and nCBC [25], and since eIF4E was not detected in immunoprecipitation assays (data not shown), the CERKL-mRNA association is probably direct. However, we cannot exclude an interaction with a yet unidentified component of the 5′cap structure. In addition, CERKL was found to bind to proteins of the translation machinery, such as PABP, eIF3B, HSP70 and the ribosomal protein RPS3, all of which are found in SGs and in other mRNPs, such as neuronal and transport granules. Except for eIF3B, the interaction of CERKL with these proteins was mRNA-dependent, mostly forming compact RNPs. Interestingly, the CERKL-C125W mutant did not interact with eIF3B and its binding to the other proteins was not sensitive to RNase A even at high salt concentrations, indicating the absence of RNA in these complexes. This together with the restricted nuclear/cytoplasmic shutling ability shown for this mutant could explain why it is not found in Sgs. CERKL-containing compact mRNPs were found associated with microtubules and in the cytoskeletal fraction. In fact, CERKL contains a PH domain that is also present in proteins that bind to microtubules. This association could be found here in distal compartments of differentiated neural cells. These results suggest a role of this protein in the transport of the mRNAs present in these structures, although this needs to be further investigated. In this regard, it is known that a compact packaging of mRNAs into mRNPs is essential to protect the mRNAs and to guarantee their survival until translation occurs. The assembly of these mRNPs starts while the mRNA is still being transcribed. This explains the requirements of an active transcription, the nuclear import/export cycle of CERKL and why the CERKL C125W mutant is unable to form these complexes. Although we have highlighted a new cellular role of CERKL and shown that a pathological mutant lacks these features, further studies are needed to substantiate the in vivo role of CERKL variants in the retina, focusing on the relationship between the protein isoforms, their intracellular localization and the disease phenotype

Ubiquitin-mediated mechanisms of translational control

mRNAs translation to proteins constitutes an important step of cellular gene expression that is highly regulated in response to different extracellular stimuli and stress situations. The fine control of protein synthesis is carried out both qualitatively and quantitatively, depending on the cellular demand at each moment. Post-translational modifications, in turn regulated by intracellular signaling pathways, play a key role in translation regulation. Among them, ubiquitination, whose role is becoming increasingly important in the control of translation, determines a correct balance between protein synthesis and degradation. In this review we focus on the role of ubiquitination (both degradative K48-linkage type and non-degradative K63-linkage type and monoubiquitination) in eukaryotic translation, both at the pre-translational level during the biogenesis/degradation of the components of translational machinery as well as at the co-translational level under stressful conditions. We also discuss other ubiquitin-dependent regulatory mechanisms of mRNA protection and resumption of translation after stress removal, where the ubiquitination of ribosomal proteins and associated regulatory proteins play an important role in the global rhythm of translation.Peer reviewe

CERKL, a retinal disease gene,encodes an mRNA-biding protein that localizes in compact and unstranslated mRNPs associated to microtubules.

The function of CERKL (CERamide Kinase Like), a causative gene of retinitis pigmentosa and cone-rod dystrophy, still awaits characterization. To approach its cellular role we have investigated the subcellular localization and interaction partners of the full length CERKL isoform, CERKLa of 532 amino acids, in different cell lines, including a photoreceptor-derived cell line. We demonstrate that CERKLa is a main component of compact and untranslated mRNPs and that associates with other RNP complexes such as stress granules, P-bodies and polysomes. CERKLa is a protein that binds through its N-terminus to mRNAs and interacts with other mRNA-binding proteins like eIF3B, PABP, HSP70 and RPS3. Except for eIF3B, these interactions depend on the integrity of mRNAs but not of ribosomes. Interestingly, the C125W CERKLa pathological mutant does not interact with eIF3B and is absent from these complexes. Compact mRNPs containing CERKLa also associate with microtubules and are found in neurites of neural differentiated cells. These localizations had not been reported previously for any member of the retinal disorders gene family and should be considered when investigating the pathogenic mechanisms and therapeutical approaches in these diseases

CERKL interacts with components of the translation machinery and with mRNAs.

<p><b>A</b>) HEK-293T cells overexpressing Flag-tagged GFP, CERKL- WT or CERKL-C125W were lysed and Flag was immunoprecipitated as described in Materials and Methods, analyzed by SDS-PAGE in 8% (left panel) and 20% (right panel) polyacrylamide gels and silver-stained. Proteins were characterized by mass spectrometry (see Materials and Methods). The positions of the proteins are indicated by arrows, from up to down as follows: eIF3B (1), CERKL (2), alpha-tubulin (3), ß-tubulin (4) (8%polyacrylamide gel), PABP (5), HSP70 (6), CERKL (7) and RPS3 (8) (20% polyacrylamide gel, WT lane) and GFP (9) (20% polyacrylamide gel, GFP lane). <b>B</b>) Flag immunoprecipitations (FLAG IP) of protein homogenates from HEK 293T cells transfected with CERKL-Flag (WT), CERKL-C125W-Flag (CW) or, as a control, GFP-Flag (GFP). The position of CERKL and GFP, detected with anti-Flag, is shown. Specific antibodies were used to detect eIF3B, PABP, HSP70 and RPS3. <b>C</b>) EMSA of CERKL protein incubated at the indicated concentrations with biotinylated mRNAs from human retina. BSA (CONTROL) and His-MBP (0) were used as negative controls. Addition of an excess of non-biotinylated probes reduced the intensity of the shifted bands (COMPETITION). Arrowheads indicate the position of the shifted bands. <b>D</b>) Two CERKL proteins moieties, Nter (amino acids 1–256) and Cter (amino acids 252–532) and the two negative controls from <b>C</b> were analyzed by EMSA using biotinylated mRNAs from COS-7 cells. Arrowhead indicates the position of the shifted band. <b>E</b>) Flag-tagged CERKL-WT or GFP proteins transiently expressed in HEK 293T cells were purified by immunoprecipitation, eluted with Flag peptide and then incubated with m⁷-GTP bound to Sepharose 4B beads (M7-GTP). Proteins bound to these beads were analyzed by Western blot with anti-Flag. As control, unconjugated Sepharose 4B beads (Seph) were used. Only CERKL-WT-Flag, but not GFP-Flag, binds to m⁷-GTP-Sepharose.</p

CERKL interacts with microtubules.

<p><b>A</b>) COS-7 cells overexpressing CERKL were fixed with 2% paraformaldehyde and the localizations of CERKL (HA) and ß-tubulin were compared by immunofluorescence. As control, cells were treated with 1 µg/ml colchicine (COL) for 2 h to disrupt microtubules. Bar: 10 µm. <b>B</b>) CERKL colocalizes with microtubule-related structures. COS-7 transfected cells were fixed with 2% paraformaldehyde and the localization of CERKL (HA) was compared by immunofluorescence with that of acetyl-α-tubulin (upper panels) and the centrosomal protein pericentrin (lower panels, arrowhead) using specific antibodies. Images at higher magnification of the rectangles in the upper row are shown below. All bars: 10 µm. <b>C</b>) Immunoblot of alpha and ß-tubulin, eIF3B, PABP, HSP70 and RPS3 proteins co-immunoprecipitating with CERKL-Flag or GFP-Flag in the presence (+) or not (−) of 1 µg/ml colchicine (COL), as indicated. Bottom, CERKL and GFP in the various lanes using anti-Flag. <b>D</b>) COS-7 cells transfected with CERKL-HA were fixed with 2% paraformaldehyde and CERKL (HA) was localized by immunofluorescence. Images at higher magnification of the rectangles are shown on the right. Arrowheads indicate the particles containing CERKL. Bars: 10 µm (original image) and 2 µm (zoom).</p

CERKL localizes to SGs and P-bodies.

<p><b>A</b> and <b>B</b>) Colocalization of CERKL (HA) with three markers of SGs (PABP and eIF4E, detected with specific antibodies described in the Materials and Methods section), and the mRNAs poly(A) tail, detected with oligo(dT) FISH (upper, middle and lower panels, respectively) in COS-7 cells overexpressing CERKL-HA, either untreated (<b>A</b>) or incubated with 500 µM sodium arsenite for 30 min (<b>B</b>). <b>C</b>) The localizations of PABP and CERKL (HA) in transfected 661W mouse photoreceptor derived cells were compared by immunofluorescence. Images at higher magnification of the rectangles in the upper row are shown below. <b>D</b>) COS-7 cells transfected with CERKL-HA were untreated (MOCK) or treated for 30 min with 100 µg/mL cycloheximide (CHX) and the localizations by immunofluorescence of PABP and CERKL (HA) were compared. <b>E</b>) COS-7 cells were transfected with CERKL-HA and treated with sodium arsenite as in <b>B</b>. The localizations by immunofluorescence of CERKL (HA) and the P-body marker EDC4 were compared. Images at higher magnification of the rectangles in the upper row are shown below. Arrowheads indicate various colocalizations. All bars: 10 µm.</p

Formation of SGs by overexpression of CERKL requires the nuclear import/export of the protein.

<p><b>A</b>) COS-7 cells overexpressing CERKL-HA were mock-treated (upper row) or treated for 2 h with 40 nM leptomycin B (LeptB), 1 µg/mL actinomycin D (ActD) or 100 µg/mL alpha-amanitin (α-Aman). Then, the localizations of CERKL (HA) and PABP (marker of SGs) were investigated by immunofluorescence. <b>B</b>) COS-7 cells overexpressing the CERKL-C125W mutant were mock-treated or treated with 500 µM SA for 30 min and the localizations of the mutant CERKL (CERKL C125W-HA) and PABP (upper panels) or eIF4E (lower panels) were investigated by immunofluorescence. Images at higher magnification of the rectangles are shown on the right. All bars: 10 µm.</p

CERKL localization in differentiated SH-SY5Y neural cells.

<p><b>A</b>) SH-SY5Y cells were transfected with CERKL-HA and after 48 h cells were differentiated with 10 µM retinoic acid. The distribution of CERKL and acetyl-α-tubulin were compared after 36 h by immunofluorescence. Images at higher magnification of the rectangles in the upper row are shown below. <b>B</b>–<b>D</b>) SH-SY5Y were transfected and differentiated as in <b>A</b>. Images show colocalization by immunofluorescence of CERKL (HA) with eIF3B (<b>B</b>), PABP (<b>C</b>) and RPS3 (<b>D</b>) in differentiated SH-SY5Y cells. Images at higher magnification of the rectangles in the upper row are shown below. <b>E</b>) Colocalization of CERKL-WT and CERKL-C125W (HA) with RNA (propidium iodide staining, PI) in differentiated SH-SY5Y cells. Arrowheads show colocalization in particulated structures. The scatter diagrams below show colocalization dots. Bars: 10 µm (original images) and 5 µm (zoom).</p