Perlman syndrome nuclease DIS3L2 controls cytoplasmic non-coding RNAs and provides surveillance pathway for maturing snRNAs

The exosome-independent exoribonuclease DIS3L2 is mutated in Perlman syndrome. Here, we used extensive global transcriptomic and targeted biochemical analyses to identify novel DIS3L2 substrates in human cells. We show that DIS3L2 regulates pol II transcripts, comprising selected canonical and histone-coding mRNAs, and a novel FTL short RNA from the ferritin mRNA 5� UTR. Importantly, DIS3L2 contributes to surveillance of maturing snRNAs during their cytoplasmic processing. Among pol III transcripts, DIS3L2 particularly targets vault and Y RNAs and an Alu-like element BC200 RNA, but not Alu repeats, which are removed by exosome-associated DIS3. Using 3� RACE-Seq, we demonstrate that all novel DIS3L2 substrates are uridylated in vivo by TUT4/TUT7 poly(U) polymerases. Uridylationdependent DIS3L2-mediated decay can be recapitulated in vitro, thus reinforcing the tight cooperation between DIS3L2 and TUTases. Together these results indicate that catalytically inactive DIS3L2, characteristic of Perlman syndrome, can lead to deregulation of its target RNAs to disturb transcriptome homeostasis

Uridylation by TUT4/7 Restricts Retrotransposition of Human LINE-1s

LINE-1 retrotransposition is tightly restricted by layers of regulatory control, with epigenetic pathways being the best characterized. Looking at post-transcriptional regulation, we now show that LINE-1 mRNA 3′ ends are pervasively uridylated in various human cellular models and in mouse testes. TUT4 and TUT7 uridyltransferases catalyze the modification and function in cooperation with the helicase/RNPase MOV10 to counteract the RNA chaperone activity of the L1-ORF1p retrotransposon protein. Uridylation potently restricts LINE-1 retrotransposition by a multilayer mechanism depending on differential subcellular localization of the uridyltransferases. We propose that uridine residues added by TUT7 in the cytoplasm inhibit initiation of reverse transcription of LINE-1 mRNAs once they are reimported to the nucleus, whereas uridylation by TUT4, which is enriched in cytoplasmic foci, destabilizes mRNAs. These results provide a model for the post-transcriptional restriction of LINE-1, revealing a key physiological role for TUT4/7-mediated uridylation in maintaining genome stability

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

Original files of the high content imaging flow cytometry

<p>These are original files which were used to generate figures' panels</p&gt

Structural analysis of mtEXO mitochondrial RNA degradosome reveals tight coupling of nuclease and helicase components

The mitochondrial RNA degradosome (mtEXO) plays an essential role in the regulation of mitochondrial gene expression and is composed of the 3′-to-5′ exoribonuclease Dss1 and the helicase Suv3. Here the authors present the RNA bound mtEXO crystal structure and give insights into its mechanism

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

Comparison of gene expression inducers.

<p>(A-D) Cells were treated with different concentrations of tetracycline or doxycycline and gene expression was monitored by western blot (A: anti-EGFP, B: anti-THOC5 antibodies, Ponceau S staining of the membrane was performed as a loading control) or flow cytometry (C, D: EGFP fluorescence). (D) Quantitative representation of data shown in panel C. Data are represented as mean ± SD (n = 3). (E) Analysis of the kinetics of expression of the indicated transgenes. Cells were treated with tetracycline, collected after indicated time and analyzed by flow cytometry. Mean fluorescent intensity of EGFP positive cells is shown (mean ± SD, n = 3).</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