The comprehensive interactomes of human adenosine RNA methyltransferases and demethylases reveal distinct functional and regulatory features

N6-methyladenosine (m(6)A) and N6,2 '-O-dimethyladenosine (m(6)Am) are two abundant modifications found in mRNAs and ncRNAs that can regulate multiple aspects of RNA biology. They function mainly by regulating interactions with specific RNA-binding proteins. Both modifications are linked to development, disease and stress response. To date, three methyltransferases and two demethylases have been identified that modify adenosines in mammalian mRNAs. Here, we present a comprehensive analysis of the interactomes of these enzymes. PCIF1 protein network comprises mostly factors involved in nascent RNA synthesis by RNA polymerase II, whereas ALKBH5 is closely linked with most aspects of pre-mRNA processing and mRNA export to the cytoplasm. METTL16 resides in subcellular compartments co-inhabited by several other RNA modifiers and processing factors. FTO interactome positions this demethylase at a crossroad between RNA transcription, RNA processing and DNA replication and repair. Altogether, these enzymes share limited spatial interactomes, pointing to specific molecular mechanisms of their regulation.Peer reviewe

Actin and myosin in transcriptional and post-transcriptional control of gene expression

Actin plays a key role in basal gene regulation. In the past decade actin has been found to be a component of chromatin remodeling complexes, ribonucleoprotein particles and to associate with all eukaryotic RNA polymerases. Based on the above discoveries the main objective of this thesis has been to elucidate some of the molecular mechanisms through which nuclear actin controls synthesis and processing of RNA transcripts. In mammals, elongation of pre-mRNA transcripts is regulated by the interaction between actin and the heterogeneous nuclear ribonucleoprotein hnRNP U. In Paper I we investigated the molecular mechanisms underlying their cooperative function. We discovered that actin and hnRNP U interact with the phosphorylated RNA polymerase II carboxy-terminal domain to recruit the histone acetyl transferase PCAF to active genes. This mechanism is required to establish permissive chromatin for efficient transcription elongation. There is emerging evidence that changes in the polymerization state of nuclear actin are important for gene regulation. Along these lines, in Paper II we found that nuclear actin dynamics is necessary for RNA polymerase II mediated transcription. We show that the F-actin severing protein cofilin-1 maintains the pool of monomeric actin to be fed into growing actin polymers and this mechanism is specifically required for elongation of pre-mRNA. Altogether these findings suggest actin polymerization occurs to facilitate migration of elongating RNA polymerase II along active genes. Evidence that the interaction between actin and nuclear myosin 1 (NM1) is important for RNA polymerase I transcription elongation led us to investigate their potential synergy in post-transcriptional control of rRNA biogenesis. In Paper III we found that in nucleoli NM1 associates with rRNA, NM1 becomes incorporated into newly synthesized ribosomal subunits and cooperates with actin for their maturation. We also found that rRNA-associated NM1 interacts with the export receptor CRM1 and the RNA binding nucleoporin Nup153 at the basket of the nuclear pore complex (NPC). We propose that NM1 accompanies newly assembled export-competent ribosomal subunits from nucleolus to NPC, thus modulating both their maturation and export

The F-actin severing protein cofilin-1 is required for RNA polymerase II transcription elongation

In mammals actin contributes to transcription elongation by facilitating establishment of permissive chromatin. Here we report that the F-actin severing protein cofilin-1 is part of the same complex with actin and phosphorylated RNA polymerase (pol) II. In chromatin immunoprecipitation assays cofilin-1 was found selectively associated with transcribed regions of active genes, its occupancy being influenced by the polymerization state of actin. Cofilin-1 gene silencing led to a drop in FUrd incorporation into nascent transcripts. In cofilin-1 silenced cells chromatin immunoprecipitations showed that active genes were devoid of actin, phosphorylated pol II and displayed low histone H3 acetylation levels on K9. These findings suggest that cofilin-1 plays a major role in pol II transcription, facilitating association of elongating pol II and actin with active genes. We speculate that cofilin-1 performs its function in pol II transcription by regulating polymerization of gene-associated actin

The EJC Binding and Dissociating Activity of PYM Is Regulated in Drosophila

International audienc

Regulation of PYM function by its domains.

<p>(A) Lysates of S2 cells co-expressing HA-eIF4AIII and GFP (control) or GFP-tagged PYM proteins (as indicated at the top of the panel) were immunoprecipitated using protein G (Mock), GFP-Trap (α-GFP IP) and anti-HA (α -HA IP) beads. Input panel shows 1.6% of the extracts and the bound fractions are shown in separate panels. The antibodies used for western analysis are indicated on the right of the panel. The anti-PYM antibody was not used for detection of PYM-GFP proteins due its preferential detection of the <i>Dm</i>PYM C-terminus (data not shown). Arrow and arrowhead indicate endogenous and HA-tagged eIF4AIII proteins, respectively. (B) Lysates of S2 cells expressing GFP (control) or GFP-tagged FL-, ΔC-, or N-PYM proteins were subjected to immunoprecipitation using protein G (Mock) or GFP-Trap (GFP IP) beads under native and DSP cross-linked conditions. 1.6% and 0.32% of inputs utilised in IPs were loaded in lanes 1, 7, 13, 19 and lanes 2, 8, 14, 20, respectively. Bound fractions (20%) were loaded in lanes 4, 10, 16, 22, and the corresponding 5× and 20× dilutions in lanes 5, 11, 17, 23 and lanes 6, 12, 18, 24 respectively. Lanes 3, 9, 15 and 21 contain 20% of the mock IP precipitates. Antibodies utilized for the western blot analysis are indicated at the right of the panels. (C) CoIP efficiencies of Mago and Y14 with GFP (control) or GFP-tagged FL-, ΔC- and N-PYM proteins under native (left panel) and DSP cross-linked (right panel) conditions. Mago and Y14 coIP efficiency is defined as a percentage of measured GFP enrichment in the corresponding GFP IPs. Plotted bar values represent the mean of two biological and four technical replicates.</p

Over-expression of the N-terminal of <i>Dm</i>PYM affects <i>oskar</i> transport.

<p>(A–F) Distribution of Staufen (red, left panel) and Oskar (greyscale, right panel) proteins as revealed by immunostaining of wild-type stage 9 egg-chambers expressing GFP-tagged PYM transgenes as indicated to the right of the panel. DAPI is in cyan. Scale bar 25 µm. (G–I) Fluorescent <i>in situ</i> hybridization and immunostaining showing the distribution pattern of <i>oskar</i> mRNA (red; left panel) and Oskar protein (greyscale; right panel) in <i>osk^A87</i>/+ egg-chambers expressing FLAG-FL-PYM (G), FLAG-ΔC-PYM (H), or FLAG-ΔN-PYM (I). <i>oskar</i> mRNA was detected using a <i>oskar</i> 3′UTR probe. DAPI is shown in cyan. Scale bar 25 µm. (J and K) Immunoprecipitation from cytoplasmic extracts from <i>osk^A87/+</i> ovaries expressing FLAG-tagged PYM proteins using mouse anti-FLAG antibody. The protein precipitates from <i>osk^A87/+</i> (J, lanes 3 and 5), and <i>osk^A87/+</i> expressing FL-PYM (J, lanes 4 and 6), ΔN-PYM (K, lanes 4 and 6), or ΔC-PYM (K, lanes 3 and 5) ovarian extracts were western blotted and probed with the antibodies indicated at the right of the panels. The inputs (1%) are shown in lanes 1 and 2 of the panels. Endogenous PYM is indicated by an arrow. The electrophoretic mobility of the FLAG-ΔN- and ΔC-PYM proteins in K is indistinguishable from that of the endogenous PYM protein. An asterisk denotes the IgG heavy chain.</p

Endogenous <i>Dm</i>PYM interacts with Y14 and Mago but not with ribosomes.

<p>(A) Immunoprecipitation using anti-HA (lane 2) and anti-PYM (lane 3) antibody was performed using wild-type ovarian extracts. The precipitated proteins were analyzed by western blotting and stained with the antibodies indicated at the right of the panel. Lane 4 shows the anti-PYM precipitate from an extract treated with RNase. Input (1%) is shown in lane 1. (B) Sucrose cushion centrifugation of wild-type cytoplasmic ovarian extract. The input (lane 1; 50%), supernatant (lane 2) and pellet (lane 3) fractions were processed for western blot analysis and stained with the antibodies indicated at the right of the panel.</p

PYM is a non-essential gene in <i>Drosophila</i>.

<p>(A) Schematic diagram showing the genomic organization of <i>pym</i> (<i>wibg</i>, shown in blue) relative to the <i>bgcn</i> gene (show in green) in the right arm of the second chromosome (2R). The centromere is to the left and the telomere is at the right. Open boxes and interconnecting lines represent exons and introns, respectively. The 5′UTRs are shown as filled black boxes. The insertion site of the P-element, <i>P{lacW}wibg^SH1616</i> is depicted as a triangle. (B and C) Western blot analysis of <i>Drosophila</i> adult (B) and ovary (C) extracts shows the absence of PYM protein from <i>pym</i> null flies (B, lanes 3 and 4; C, lane 2) as compared to the wild-type (WT; B, lanes 1 and 2; C, lane 1). The antibodies used for staining are indicated on the right of the panel. S = short Oskar, L = long Oskar, KHC = kinesin heavy chain. (D) Fluorescent <i>in situ</i> hybridization coupled with immunostaining of wild-type (WT, upper panel) and <i>pym</i> null (lower panel) egg-chambers during stages 8 and 9 of oogenesis. <i>oskar</i> mRNA is detected with a 3′UTR probe (red) while anti-Oskar staining is shown in greyscale. DAPI is in cyan. Scale bar 25 µm.</p

Ectopic <i>Dm</i>PYM disassembles EJC on the mRNAs.

<p>(A) Cytoplasmic ovarian extract from wild-type (WT), <i>osk^A87/+</i>, and <i>osk^A87/+</i> flies expressing FLAG- or GFP-tagged FL-PYM were immunoprecipitated using anti-eIF4AIII antibody (lanes 9–12) or rabbit IgG (lanes 5–8). The inputs (1%, lanes 1–4) and the bound protein samples were analyzed by western blotting using antibodies indicated at the right of the panel. An asterisk indicates the heavy chain of IgG. (B) <i>In vitro</i> splicing of ³²P-labelled <i>oskE1E2(iftz)</i>(lanes 2–11) and <i>oskE1E2(intronless)</i> (lanes 2′–11′) RNAs was carried out using embryo nuclear extract for 180 min. Aliquots of the reactions were supplemented with buffer (lanes 3 and 3′), GST (0.5 µM (+, lanes 4 and 4′) and 1 µM (++, lanes 5 and 5′)), GST-FL-PYM (0.5 µM (+, lanes 6 and 6′) and 1 µM (++, lanes 7 and 7′)), GST-ΔN-PYM (0.5 µM (+, lanes 8 and 8′) and 1 µM (++, lanes 9 and 9′)) or GST-ΔC-PYM (0.5 µM (+, lanes 10 and 10′) and 1 µM (++, lanes 11 and 11′)) and incubated for 30 min. An oligonucleotide centered at −25 relative to the first splice junction of <i>oskar</i> was added to elicit RNase H cleavage (lanes 3–11, 3′–11′) and the samples were resolved by urea PAGE. The presence of RNA cleavage products (indicated by an asterisk) in lanes 6, 7, 10 and 11 suggests loss of protection from RNase H cleavage due to disassembly of the EJC. The positions of the pre-mRNA, mRNA and splicing intermediates and products are shown at the sides of the panel. (C) Top panel: Semi-quantitative RT-PCR analysis of the mRNAs (indicated on the right of the panel) obtained by immunoprecipitation using GFP-Trap beads either from <i>osk^A87/+</i> ovarian extracts or <i>osk^A87/+</i> ovaries co-expressing GFP-tagged Mago and one of the FLAG-tagged PYM constructs, as indicated at the top of the panel. Lanes 1–4 show the input samples, and lanes 5–8 show mRNAs recovered in the immunoprecipitates. Bottom panel: Western blot of the samples used for RT-PCR analysis stained with antibodies indicated at the right of the panel. The GFP-Mago panel was probed with anti-GFP antibody. 20% of the input and bound fractions from the immunoprecipitate was used for western analysis.</p