mRNA secondary structure.

<p>5´UTR sequences of FAIM isoforms structures as shown by the output of the RNAStructure web server (<a href="http://rna.urmc.rochester.edu/RNAstructureWeb/Servers/Predict1/Predict1.html" target="_blank">http://rna.urmc.rochester.edu/RNAstructureWeb/Servers/Predict1/Predict1.html</a>). The optimal secondary prediction for all the sequences was obtained in dot-bracket notation with the lowest free energy structure for the input sequence. Colour annotation of the structures provides information about the confidence in the prediction of a specific pair (base paired or unpaired nucleotides). The highest probabilities are red and the lowest are purple.</p

FAIM-S_2a and FAIM-L_2a are localized in the cytoplasm and nucleus.

<p><b>A:</b> Western blot analysis using anti-FLAG to detect the presence of FAIM-S, FAIM-L, FAIM-S_2a and FAIM-L_2a in the distinct cellular compartments. Anti-calnexin was used as a marker for the membrane fraction, anti-actin as a marker of the cytosolic fraction, and anti-Tri-Methyl-Histone H3 as a marker of the nucleus. <b>B:</b> Immunofluorescence in Vero cells 24 h after transfection with pcDNA3-GFP containing the extra-long isoforms. Anti-calnexin (reticular protein), Mitotracker (mitochondrial marker) and Hoechst (nuclei staining) were used to examine the co-localization of FAIM isoforms. Scale bars 10 <b>μ</b>m.</p

List of specific primers for each exon used for RT-PCR.

<p>List of specific primers for each exon used for RT-PCR.</p

Treatment with Actinomycin D (ActD).

<p>SH-SY5Y cells were treated with ActD for a range of times (3, 6, 9 and 12 h). <b>A:</b> The half-life of mRNA was measured by treating cells with ActD (5 μg/ml) and collecting total RNA at the times shown, whereupon the levels of <i>FAIM</i> mRNA and <i>18S mRNA</i> (a stable, housekeeping control mRNA) were measured by RT–qPCR analysis. mRNA half-life was calculated as the time needed to reduce transcript levels to half (50%, discontinuous line) of their initial abundance at time 0. <b>B:</b> Number of cycles needed to detect similar size product of FAIM-S, FAIM-L, FAIM-S_2a and FAIM-L_2a by qPCR (SybrGreen) using the following pairs of primers: (1bF2/3R for FAIM-S; 2bF/3R for FAIM-L; 2aF/2a3R for FAIM-S_2a and 2aF/2bR for FAIM-L_2a).</p

Isoforms expression in cell lines.

<p><b>A:</b> SH-SY5Y cells were transfected with the pCDNA3-FLAG-FAIM-S, pCDNA3-FLAG-FAIM-S_2a, pCDNA3-FLAG-FAIM-L or pCDNA3-FLAG-FAIM-L_2a vector. At a range of time points, cells were harvested and protein expression was assessed by western blot using an anti-FLAG antibody (dilution 1:20000). <b>B</b>: PC12 cells were transfected with the isoform vectors (above mentioned) and treated with MG-132 (2.5 μM). Cell extracts were then resolved by western blot analysis, and FAIM expression was measured using an anti-FLAG antibody (dilution 1:20000). <b>C:</b> HEK293T cells transfected with pcDNA3-FLAG-FAIM-L, pcDNA3-FLAG-FAIM-S, pcDNA3-FLAG-FAIM-L-2a or pcDNA3-FLAG-FAIM-S-2a vector were lysed, and protein extracts were analyzed by western blot. An anti-FAIM-L (anti-2b FAIM, specific for neuronal exon 2b) and anti-FAIM (that recognizes the common part of the isoforms) were used. Anti-tubulin was used as a loading control. Two different exposures of the film are shown in order to facilitate observation of the bands of all isoforms. DIV: days <i>in vitro</i> (n = 3).</p

Thermodynamic stability of the secondary structure of the 5´UTR.

<p>Thermodynamic stability of the secondary structure of the 5´UTR.</p

FAIM-S_2a and FAIM-L_2a are expressed in human cell lines and human tissues.

<p><b>A:</b> Schematic representation of the primers used for RT-PCR and the localization in each exon. The 5´ and 3´UTRs are represented in gray, and CDS regions in black. Length of exons is indicated in base pairs (bp). TSS: Transcriptional start site, TSS1: TSS of FAIM-S_2a and FAIM-L_2a; TSS2: TSS of FAIM-L; TSS3: TSS of FAIM-S. <b>B:</b> RT-PCR analysis of exon 2a in human cell lines using a 2aF/6R primer combination (the 2aF primer is common to exon 2a in FAIM-L_2a and FAIM-S_2a). Bands of 657 and 597 bp were detected in the neuroblastoma cell line (SH-SY5Y) corresponding to the predicted size of FAIM-L_2a and FAIM-S_2a, respectively. In non-neuronal derived cell lines, namely HEK293T and SK-N-AS, only one band of FAIM-S_2a was detected. <b>C:</b> RT-PCR analysis of all the four isoforms in the fetal cortex, fetal and adult testes and placenta using specific primers for exon 2a (2aF/6R) and 1b (1bF/3R). <b>D:</b> RT-PCR analysis of exon 1b in HEK293T, SH-SY5Y and SK-N-AS cell lines. Primers used: 1bF/3R or 1bF/2aR. Only one band was observed in SH-SY5Y cells, but FAIM-L_2a and FAIM-S_2a were estimated to have a band of 470 bp. <b>E:</b> mRNA amplification of FAIM-S, FAIM-S_2a and FAIM-L_2a using a primer in the region of exon 1a (1aF/3R; 1aF/2a-3R; 1aF/2bR). Negative control (C(-)) was performed with water instead of cDNA. 100 bp DNA Ladder Plus was used to determine the size of each DNA band. L27 was used as an internal control. The primers used are indicated below the agarose gel.</p

Overexpression of nSR100 induce FAIM-L_2a and FAIM-L in HEK293T cells.

<p><b>A:</b> nSR100 transcript was amplified by RT-PCR. In SH-SY5Y cells, a band of 420 bp was detected. <b>B:</b> RT-PCR in HEK293T cells after transient transfection with nSR100 using Lipofectamine 2000®. Transcripts of FAIM-L and FAIM-L_2a were observed at 732 bp and 657 bp bands. L27 was used as an internal control in all PCRs. <b>C:</b> Western blot analysis using anti-FAIM in HEK293T cells after transfection with nSR100 vector (pLD_hsnSR100). A band of 23 kDa (FAIM-L) was detected in nSR100 transfection conditions. As a negative control, we used an empty vector (n = 3).</p