Nuclear and cytosolic hCLE-associated proteins.

<p>Table includes proteins where at least 2 non-redundant peptides were identified, with a FDR <1% at peptide level that are present exclusively in hCLE purified samples or have at least 3-fold enrichment of total number of confidently identified peptides in hCLE purified fractions compared to the control (ctr.) samples (marked with asterisk). Their molecular weight, the total number of confidently identified peptides and the number of non-redundant ones, as well as the protein Mascot scores, is shown.</p

Nuclear and cytosolic hCLE-associated proteins.

<p>Table includes proteins where at least 2 non-redundant peptides were identified, with a FDR <1% at peptide level that are present exclusively in hCLE purified samples or have at least 3-fold enrichment of total number of confidently identified peptides in hCLE purified fractions compared to the control (ctr.) samples (marked with asterisk). Their molecular weight, the total number of confidently identified peptides and the number of non-redundant ones, as well as the protein Mascot scores, is shown.</p

hCLE/C14orf166 Associates with DDX1-HSPC117-FAM98B in a Novel Transcription-Dependent Shuttling RNA-Transporting Complex

<div><p>hCLE/C14orf166 is a nuclear and cytoplasmic protein that interacts with the RNAP II, modulates nuclear RNA metabolism and is present in cytoplasmic RNA granules involved in localized translation. Here we have studied whether hCLE shares common interactors in the nucleus and the cytosol, which could shed light on its participation in the sequential phases of RNA metabolism. Nuclear and cytoplasmic purified hCLE-associated factors were identified and proteins involved in mRNA metabolism, motor-related proteins, cytoskeletal and translation-related factors were found. Purified hCLE complexes also contain RNAs and as expected some hCLE-interacting proteins (DDX1, HSPC117, FAM98B) were found both in the nucleus and the cytoplasm. Moreover, endogenous hCLE fractionates in protein complexes together with DDX1, HSPC117 and FAM98B and silencing of hCLE down-regulates their nuclear and cytosolic accumulation levels. Using a photoactivatable hCLE-GFP protein, nuclear import and export of hCLE was observed indicating that hCLE is a shuttling protein. Interestingly, hCLE nuclear import required active transcription, as did the import of DDX1, HSPC117 and FAM98B proteins. The data indicate that hCLE probably as a complex with DDX1, HSPC117 and FAM98B shuttles between the nucleus and the cytoplasm transporting RNAs suggesting that this complex has a prominent role on nuclear and cytoplasmic RNA fate.</p></div

Identification of hCLE as monomer and dimer in denaturing gel conditions.

<p><b><i>AC</i></b><b>:</b> Accession Number of the top protein from NCBInr protein Database (non-identical NCBI protein database).</p><p><b><i>MW (Mr):</i></b> Nominal molecular weight of each protein.</p><p><b><i>pI:</i></b> Calculated pI value.</p><p><b><i>Pept (MS[MSMS]):</i></b> number of matched peptides from the top scoring protein in peptide mass fingerprinting and number of MS/MS spectra that were matched to this protein.</p><p><b><i>Score:</i></b> Mascot protein score. This number reflects the combined scores of all observed mass spectra that can be matched to amino acid sequences within that protein. A higher score indicates a more confident match.</p><p><b><i>Seq Cov:</i></b> Percentage of the database protein sequence covered by matching peptides.</p

hCLE nuclear import is transcription dependent.

<p>Cultured HEK293T cells were transfected with the phCLE-PAGFP (photoactivatable GFP) plasmid and 24 h post-transfection they were treated with Actinomycin D during a 10 min pulse, washed and used for live cell microscopy. (A), Photoactivation was applied in the cytosol to visualize hCLE import. (B), Photoactivation was applied in the nucleus to visualize hCLE export. A dotted line marking the boundary of the nucleus is included in the last panels.</p

hCLE forms dimers that are resistant to denaturing conditions.

<p>(A); Western blot against hCLE of total extracts of HEK293T cells using denaturing (left) or native conditions (right). (B); Diagram showing the scheme of the fusion protein hCLE-TAP with its tags for affinity purification. (C); left, HEK293T cells were transfected with the hCLE-TAP expressing plasmid and total cell extract was used for affinity purification, a sample of the purified protein was analyzed by silver staining in SDS-PAGE gels. Right; The hCLE-CBC purified protein was dialyzed and analyzed in non-denaturing gels followed by Western blot detection. (D). The hCLE-CBD purified protein was subjected to silver staining and bands corresponding to different sizes were excised and analyzed by MS-MS technique. The lines denote hCLE identification by MS-MS technique. (*), denotes hCLE monomer and (**) hCLE dimer.</p

Transcription inhibition increases the cytosolic accumulation of hCLE, DDX1, HSPC117 and FAM98B.

<p>Cultured HEK293T cells were incubated in the absence or the presence of Actinomycin D (Act. D) during 1 h. Nuclear and cytoslic extracts were prepared and used for Western blot assays to detect the indicated proteins. (N); nuclear fraction, (C), cytosolic fraction. RNAP II and α-actin were used as markers for subcellular fractionation.</p

Transcription inhibition retains DDX1 and HSPC117 in the cytoplasm.

<p>Cultured HEK293T cells were incubated in the absence (Control) or the presence of Actynomicin D (Act. D) during 30 min, washed, fixed and processed for immunofluorescence using antibodies anti-hCLE, DDX1, HSPC117 and DAPI.</p

hCLE modulates the expression of hCLE-interacting proteins.

<p>(A); HEK293T cells were infected with lentiviruses expressing a control sequence (Ct) or specific sequences for hCLE silencing (siCLE.1 and si-CLE.2) and 4 days post-infection total cell extracts were used for Western blot against the indicated proteins. (B); HEK293T cells were infected with lentiviruses expressing a control sequence (Ct) or a specific sequence for hCLE silencing (siCLE.1). 5 days post-infection, the cells were left untransfected (-) or transfected (+) with a plasmid expressing a hCLE gene with 3 silent mutations that avoids its silencing (ns.hCLE). 48<b> </b>h post-transfection, nuclear and cytoplasmic fractions were prepared and used for Western blot detection of the indicated proteins.</p

Nuclear and cytosolic hCLE-associated proteins.

<p>Table includes proteins where 1 non-redundant peptides were identified, with a FDR <1% at peptide level that are present exclusively ihCLE purified samples. Their molecular weight, the total number of confidently identified peptides and the number of non-redundant ones, as well as the protein Mascot scores, is shown.</p