Two novel C-terminal frameshift mutations in the β-globin gene lead to rapid mRNA decay

BACKGROUND: The thalassemia syndromes are classified according to the globin chain or chains whose production is affected. β-thalassemias are caused by point mutations or, more rarely, deletions or insertions of a few nucleotides in the β-globin gene or its immediate flanking sequences. These mutations interfere with the gene function either at the transcriptional, translational or posttranslational level. METHODS: Two cases of Polish patients with hereditary hemolytic anemia suspected of thalassemia were studied. DNA sequencing and mRNA quantification were performed. Stable human cell lines which express wild-type HBB and mutated versions were used to verify that detected mutation are responsible for mRNA degradation. RESULTS: We identified two different frameshift mutations positioned in the third exon of HBB. Both patients harboring these mutations present the clinical phenotype of thalassemia intermedia and showed dominant pattern of inheritance. In both cases the mutations do not generate premature stop codon. Instead, slightly longer protein with unnatural C-terminus could be produced. Interestingly, although detected mutations are not expected to induce NMD, the mutant version of mRNA is not detectable. Restoring of the open reading frame brought back the RNA to that of the wild-type level. CONCLUSION: Our results show that a lack of natural stop codon due to the frameshift in exon 3 of β-globin gene causes rapid degradation of its mRNA and indicate existence of novel surveillance pathway

Versatile approach for functional analysis of human proteins and efficient stable cell line generation using FLP-mediated recombination system

Deciphering a function of a given protein requires investigating various biological aspects. Usually, the protein of interest is expressed with a fusion tag that aids or allows subsequent analyses. Additionally, downregulation or inactivation of the studied gene enables functional studies. Development of the CRISPR/Cas9 methodology opened many possibilities but in many cases it is restricted to non-essential genes. Recombinase-dependent gene integration methods, like the Flp-In system, are very good alternatives. The system is widely used in different research areas, which calls for the existence of compatible vectors and efficient protocols that ensure straightforward DNA cloning and generation of stable cell lines. We have created and validated a robust series of 52 vectors for streamlined generation of stable mammalian cell lines using the FLP recombinase-based methodology. Using the sequence-independent DNA cloning method all constructs for a given coding-sequence can be made with just three universal PCR primers. Our collection allows tetracycline-inducible expression of proteins with various tags suitable for protein localization, FRET, bimolecular fluorescence complementation (BiFC), protein dynamics studies (FRAP), co-immunoprecipitation, the RNA tethering assay and cell sorting. Some of the vectors contain a bidirectional promoter for concomitant expression of miRNA and mRNA, so that a gene can be silenced and its product replaced by a mutated miRNA-insensitive version. Our toolkit and protocols have allowed us to create more than 500 constructs with ease. We demonstrate the efficacy of our vectors by creating stable cell lines with various tagged proteins (numatrin, fibrillarin, coilin, centrin, THOC5, PCNA). We have analysed transgene expression over time to provide a guideline for future experiments and compared the effectiveness of commonly used inducers for tetracycline-responsive promoters. As proof of concept we examined the role of the exoribonuclease XRN2 in transcription termination by RNAseq

Proteomic Analyses Reveal the Role of Alpha-2-Macroglobulin in Canine Osteosarcoma Cell Migration

Canine osteosarcoma (OSA) is an aggressive bone neoplasia with high metastatic potential. Metastasis is the main cause of death associated with OSA, and there is no current treatment available for metastatic disease. Proteomic analyses, including matrix-assisted laser desorption/ionisation-time of flight mass spectrometry (MALDI TOF/TOF MS), are widely used to select molecular targets and identify proteins that may play a key role in primary tumours and at various steps of the metastatic cascade. The main aim of this study was to identify proteins differently expressed in canine OSA cell lines with different malignancy phenotypes (OSCA-8 and OSCA-32) compared to canine osteoblasts (CnOb). The intermediate aim of the study was to compare canine OSA cell migration capacity and assess its correlation with the malignancy phenotypes of each cell line. Using MALDI-TOF/TOF MS analyses, we identified eight proteins that were significantly differentially expressed (p ≤ 0.05) in canine OSA cell lines compared to CnOb: cilia- and flagella-associated protein 298 (CFAP298), general transcription factor II-I (GTF2I), mirror-image polydactyly gene 1 protein (MIPOL1), alpha-2 macroglobulin (A2M), phosphoglycerate mutase 1 (PGAM1), ubiquitin (UB2L6), ectodysplasin-A receptor-associated adapter protein (EDARADD), and leucine-rich-repeat-containing protein 72 (LRRC72). Using the Simple Western technique, we confirmed high A2M expression in CnOb compared to OSCA-8 and OSCA-32 cell lines (with intermediate and low A2M expression, respectively). Then, we confirmed the role of A2M in cancer cell migration by demonstrating significantly inhibited OSA cell migration by treatment with A2M (both at 10 and 30 mM concentrations after 12 and 24 h) in a wound-healing assay. This study may be the first report indicating A2M’s role in OSA cell metastasis; however, further in vitro and in vivo studies are needed to confirm its possible role as an anti-metastatic agent in this malignancy

Efficiency of CDS cloning into pKK-RNAi vectors.

<p>Efficiency of CDS cloning into pKK-RNAi vectors.</p

Involvement of XRN2 in transcription termination.

<p>(A) Flow cytometry measurement of transgenes expression after 24 hours of induction (EGFP tags XRN2, mCherry is a reporter of miRNA expression). (B) Confocal live cell imaging of EGFP tagged XRN2 and Hoechst 33342 stained nuclei. (C) Western blot analysis of XRN2 protein with anti-XRN2 antibodies. Parental 293 cells and their derivatives analyzed in panel A and B were treated with tetracycline for 72 hours and subjected to western blot. Ponceau S staining of the membrane was performed as a loading control. (D) Meta-gene analysis of transcriptional read-through in wild-type and mutant XRN2 cells. Strand-specific read densities were averaged across 250-bp genomic windows placed directly downstream of 3' ends of highly expressed (TPM > 10), spliced transcripts. The signal is normalized to the average expression detected in the last 250 nt of the analyzed transcripts (250-bp windows upstream to the expected termination site). The shaded part of the graph marks transcripts downstream of transcription termination site (products of transcriptional read-through). It is important to note that lines representing RNA steady-state levels overlay in the part of the graph which correspond to RNAs originating from the transcription upstream of the transcription termination site. This is in contrast to the part of the graph which represent RNA resulting from the unsuccessful transcription termination (shaded part of the graph).</p

SLIC-based DNA cloning strategy.

<p>See main text for detailed description. RE–restriction enzymes used for vector linearization. These are BshTI and NheI in our protocol for universal SLIC. EGFP is an example of tag that can be used. A detailed protocol for the SLIC procedure can be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0194887#pone.0194887.s007" target="_blank">S2 Supporting Information</a>.</p

Efficiency of the miRNA cassette subcloning into pKK-RNAi vectors.

<p>Efficiency of the miRNA cassette subcloning into pKK-RNAi vectors.</p

Efficiency of stable cell line generation.

<p>(A) Influence of plasmid quantity and selection stringency on the number of colonies obtained following stable transfection of 293 Flp-In T-REx cells. 1.0 μg of pOG44 was mixed with the indicated amounts of pcDNA5/FRT/TO and used for transfection. Cells were selected by treatment with the indicated concentration of hygromycin B and constant concentration of blasticidin S (10 μg/ml). Colonies were stained with crystal violet. (B) Comparison of stable transfection efficiency with pcDNA5/FRT/TO or its pKK derivatives. Cells were transfected with 300 ng of indicated plasmids and 1.0 μg of pOG44 and subjected to selection with hygromycin B (50 μg/ml) and blasticidin S (10 μg/ml).</p