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

Human mitochondrial RNA turnover caught in flagranti: involvement of hSuv3p helicase in RNA surveillance

The mechanism of human mitochondrial RNA turnover and surveillance is still a matter of debate. We have obtained a cellular model for studying the role of hSuv3p helicase in human mitochondria. Expression of a dominant-negative mutant of the hSUV3 gene which encodes a protein with no ATPase or helicase activity results in perturbations of mtRNA metabolism and enables to study the processing and degradation intermediates which otherwise are difficult to detect because of their short half-lives. The hSuv3p activity was found to be necessary in the regulation of stability of mature, properly formed mRNAs and for removal of the noncoding processing intermediates transcribed from both H and L-strands, including mirror RNAs which represent antisense RNAs transcribed from the opposite DNA strand. Lack of hSuv3p function also resulted in accumulation of aberrant RNA species, molecules with extended poly(A) tails and degradation intermediates truncated predominantly at their 3′-ends. Moreover, we present data indicating that hSuv3p co-purifies with PNPase; this may suggest participation of both proteins in mtRNA metabolism

Controlling the mitochondrial antisense – role of the SUV3-PNPase complex and its co-factor GRSF1 in mitochondrial RNA surveillance

Transcription of the human mitochondrial genome produces a vast amount of non-coding antisense RNAs. These RNA species can form G-quadraplexes (G4), which affect their decay. We found that the mitochondrial degradosome, a complex of RNA helicase SUPV3L1 (best known as SUV3) and the ribonuclease PNPT1 (also known as PNPase), together with G4-melting protein GRSF1, is a key player in restricting antisense mtRNAs

Identification of a novel human mitochondrial endo-/exonuclease Ddk1/c20orf72 necessary for maintenance of proper 7S DNA levels.

Although the human mitochondrial genome has been investigated for several decades, the proteins responsible for its replication and expression, especially nucleolytic enzymes, are poorly described. Here, we characterized a novel putative PD-(D/E)XK nuclease encoded by the human C20orf72 gene named Ddk1 for its predicted catalytic residues. We show that Ddk1 is a mitochondrially localized metal-dependent DNase lacking detectable ribonuclease activity. Ddk1 degrades DNA mainly in a 3'-5' direction with a strong preference for single-stranded DNA. Interestingly, Ddk1 requires free ends for its activity and does not degrade circular substrates. In addition, when a chimeric RNA-DNA substrate is provided, Ddk1 can slide over the RNA fragment and digest DNA endonucleolytically. Although the levels of the mitochondrial DNA are unchanged on RNAi-mediated depletion of Ddk1, the mitochondrial single-stranded DNA molecule (7S DNA) accumulates. On the other hand, overexperssion of Ddk1 decreases the levels of 7S DNA, suggesting an important role of the protein in 7S DNA regulation. We propose a structural model of Ddk1 and discuss its similarity to other PD-(D/E)XK superfamily members

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

The pKK vector series.

<p>(A) Nucleotide sequences of the TEV-L and TEV-R. Translation to protein and TEV protease cleavage site are shown. Shaded letters indicate nucleotides common to both sequences. (B) Cloning sites of selected pKK vectors. Potentially useful unique restriction sites are marked. For all pKK vectors BshTI and NheI restriction enzymes are used for vector linearization before DNA cloning with the help of our universal SLIC protocol. All pKK vectors have promoters with the TetR repressor binding site. (C) Example of a pKK-BI16 vector. Map of pKK-BI16-TEV-mCherry vector and its cloning region (bottom diagram). Useful unique restriction sites are marked. The tetracycline operator sequences are present in all vectors of pKK-BI16 series, thus, transcription in both directions is regulated by the tetracycline repressor.</p