Philippe Cloutier

A Newly Uncovered Group of Distantly Related Lysine Methyltransferases Preferentially Interact with Molecular Chaperones to Regulate Their Activity

Methylation is a post-translational modification that can affect numerous features of proteins, notably cellular localization, turnover, activity, and molecular interactions. Recent genome-wide analyses have considerably extended the list of human genes encoding putative methyltransferases. Studies on protein methyltransferases have revealed that the regulatory function of methylation is not limited to epigenetics, with many non-histone substrates now being discovered. We present here our findings on a novel family of distantly related putative methyltransferases. Affinity purification coupled to mass spectrometry shows a marked preference for these proteins to associate with various chaperones. Based on the spectral data, we were able to identify methylation sites in substrates, notably trimethylation of K135 of KIN/Kin17, K561 of HSPA8/Hsc70 as well as corresponding lysine residues in other Hsp70 isoforms, and K315 of VCP/p97. All modification sites were subsequently confirmed in vitro. In the case of VCP, methylation by METTL21D was stimulated by the addition of the UBX cofactor ASPSCR1, which we show directly interacts with the methyltransferase. This stimulatory effect was lost when we used VCP mutants (R155H, R159G, and R191Q) known to cause Inclusion Body Myopathy with Pagets disease of bone and Fronto-temporal Dementia (IBMPFD) and/or familial Amyotrophic Lateral Sclerosis (ALS). Lysine 315 falls in proximity to the Walker B motif of VCPs first ATPase/D1 domain. Our results indicate that methylation of this site negatively impacts its ATPase activity. Overall, this report uncovers a new role for protein methylation as a regulatory pathway for molecular chaperones and defines a novel regulatory mechanism for the chaperone VCP, whose deregulation is causative of degenerative neuromuscular diseases.

The protein interaction network of the human transcription machinery reveals a role for the conserved GTPase RPAP4/GPN1 and microtubule assembly in nuclear import and biogenesis of RNA polymerase II.

RNA polymerase II (RNAPII), the 12-subunit enzyme that synthesizes all mRNAs and several non-coding RNAs in eukaryotes, plays a central role in cell function. Although multiple proteins are known to regulate the activity of RNAPII during transcription, little is known about the machinery that controls the fate of the enzyme before or after transcription. We used systematic protein affinity purification coupled to mass spectrometry (AP-MS) to characterize the high resolution network of protein interactions of RNAPII in the soluble fraction of human cell extracts. Our analysis revealed that many components of this network participate in RNAPII biogenesis. We show here that RNAPII-associated protein 4 (RPAP4/GPN1) shuttles between the nucleus and the cytoplasm and regulates nuclear import of POLR2A/RPB1 and POLR2B/RPB2, the two largest subunits of RNAPII. RPAP4/GPN1 is a member of a newly discovered GTPase family that contains a unique and highly conserved GPN loop motif that we show is essential, in conjunction with its GTP-binding motifs, for nuclear localization of POLR2A/RPB1 in a process that also requires microtubule assembly. A model for RNAPII biogenesis is presented.

High-resolution mapping of the protein interaction network for the human transcription machinery and affinity purification of RNA polymerase II-associated complexes

Thirty years of research on gene transcription has uncovered a myriad of factors that regulate, directly or indirectly, the activity of RNA polymerase II (RNAPII) during mRNA synthesis. Yet many regulatory factors remain to be discovered. Using protein affinity purification coupled to mass spectrometry (AP-MS), we recently unraveled a high-density interaction network formed by RNAPII and its accessory factors from the soluble fraction of human cell extracts. Validation of the dataset using a machine learning approach trained to minimize the rate of false positives and false negatives yielded a high-confidence dataset and uncovered novel interactors that regulate the RNAPII transcription machinery, including a new protein assembly we named the RNAPII-Associated Protein 3 (RPAP3) complex.

Antagonistic Effects of the SRp30c Protein and Cryptic 5 ′ Splice Sites on the Alternative Splicing of the Apoptotic Regulator Bcl-x

Alternative 5 ′ splice site selection allows Bcl-x to produce two isoforms with opposite effects on apoptosis. The pro-apoptotic Bcl-xS variant is up-regulated by ceramide and down-regulated by protein kinase C through specific cis-acting exonic elements, one of which is bound by SAP155. Splicing to the Bcl-xS 5 ′ splice site is also enforced by heterogeneous nuclear ribonucleoprotein (hnRNP) F/H proteins and by Sam68 in cooperation with hnRNP A1. Here, we have characterized exon elements that influence splicing to the 5 ′ splice site of the anti-apoptotic Bcl-xL isoform. Within a 86-nucleotide region (B3) located immediately upstream of the Bcl-xL donor site we have identified two elements (ML2 and AM2) that stimulate splicing to the Bcl-xL 5 ′ splice site. SRp30c binds to these elements and can shift splicing to the 5 ′ splice site of Bcl-xL in an ML2/AM2-dependent manner in vitro and in vivo. The B3 region also contains an element that represses the use of Bcl-xL. This element is bound by U1 small nuclear ribonucleoprotein and contains two 5 ′ splice sites that can be used when the Bcl-xL 5 ′ splice site is mutated or the ML2/AM2 elements are deleted. Conversely, mutating the cryptic 5 ′ splice sites stimulates splicing to the Bcl-xL site. Thus, SRp30c stimulates splicing to the downstream 5 ′ splice site of Bcl-xL, thereby attenuating the repressive effect of upstream U1 snRNP binding sites.

Modeling contaminants in AP-MS/MS experiments.

Identification of protein-protein interactions (PPI) by affinity purification (AP) coupled with tandem mass spectrometry (AP-MS/MS) produces large data sets with high rates of false positives. This is in part because of contamination at the AP level (due to gel contamination, nonspecific binding to the TAP columns in the context of tandem affinity purification, insufficient purification, etc.). In this paper, we introduce a Bayesian approach to identify false-positive PPIs involving contaminants in AP-MS/MS experiments. Specifically, we propose a confidence assessment algorithm (called Decontaminator) that builds a model of contaminants using a small number of representative control experiments. It then uses this model to determine whether the Mascot score of a putative prey is significantly larger than what was observed in control experiments and assigns it a p-value and a false discovery rate. We show that our method identifies contaminants better than previously used approaches and results in a set of PPIs with a larger overlap with databases of known PPIs. Our approach will thus allow improved accuracy in PPI identification while reducing the number of control experiments required.

Transcription Factor IIS Cooperates with the E3 Ligase UBR5 to Ubiquitinate the CDK9 Subunit of the Positive Transcription Elongation Factor B

Elongation of transcription by mammalian RNA polymerase II (RNAPII) is regulated by specific factors, including transcription factor IIS (TFIIS) and positive transcription elongation factor b (P-TEFb). We show that the E3 ubiquitin ligase UBR5 associates with the CDK9 subunit of positive transcription elongation factor b to mediate its polyubiquitination in human cells. TFIIS also binds UBR5 to stimulate CDK9 polyubiquitination. Co-localization of UBR5, CDK9, and TFIIS along specific regions of the γ fibrinogen (γFBG) gene indicates that a ternary complex involving these factors participates in the transcriptional regulation of this gene. In support of this notion, overexpression of TFIIS not only modifies the ubiquitination pattern of CDK9 in vivo but also increases the association of CDK9 with various regions of the γFBG gene. Notably, the TFIIS-mediated increase in CDK9 loading is obtained during both basal and activated transcription of the γFBG gene. This increased CDK9 binding is paralleled by an increase in the recruitment of RNAPII along the γFBG gene and the phosphorylation of the C-terminal domain of the RNAPII largest subunit RPB1 on Ser-2, a known target of CDK9. Together, these results identify UBR5 as a novel E3 ligase that regulates transcription and define an additional function of TFIIS in the regulation of CDK9.

Nuclear import of RNA polymerase II is coupled with nucleocytoplasmic shuttling of the RNA polymerase II-associated protein 2

The RNA polymerase II (RNAP II)-associated protein (RPAP) 2 has been discovered through its association with various subunits of RNAP II in affinity purification coupled with mass spectrometry experiments. Here, we show that RPAP2 is a mainly cytoplasmic protein that shuttles between the cytoplasm and the nucleus. RPAP2 shuttling is tightly coupled with nuclear import of RNAP II, as RPAP2 silencing provokes abnormal accumulation of RNAP II in the cytoplasmic space. Most notably, RPAP4/GPN1 silencing provokes the retention of RPAP2 in the nucleus. Our results support a model in which RPAP2 enters the nucleus in association with RNAP II and returns to the cytoplasm in association with the GTPase GPN1/RPAP4. Although binding of RNAP II to RPAP2 is mediated by an N-terminal domain (amino acids 1–170) that contains a nuclear retention domain, and binding of RPAP4/GPN1 to RPAP2 occurs through a C-terminal domain (amino acids 156–612) that has a dominant cytoplasmic localization domain. In conjunction with previously published data, our results have important implications, as they indicate that RPAP2 controls gene expression by two distinct mechanisms, one that targets RNAP II activity during transcription and the other that controls availability of RNAP II in the nucleus.

New insights into the biogenesis of nuclear RNA polymerases

More than 30 years of research on nuclear RNA polymerases (RNAP I, II, and III) has uncovered numerous factors that regulate the activity of these enzymes during the transcription reaction. However, very little is known about the machinery that regulates the fate of RNAPs before or after transcription. In particular, the mechanisms of biogenesis of the 3 nuclear RNAPs, which comprise both common and specific subunits, remains mostly uncharacterized and the proteins involved are yet to be discovered. Using protein affinity purification coupled to mass spectrometry (AP-MS), we recently unraveled a high-density interaction network formed by nuclear RNAP subunits from the soluble fraction of human cell extracts. Validation of the dataset using a machine learning approach trained to minimize the rate of false positives and false negatives yielded a high-confidence dataset and uncovered novel interactors that regulate the RNAP II transcription machinery, including a set of proteins we named the RNAP II-associated proteins (RPAPs). One of the RPAPs, RPAP3, is part of an 11-subunit complex we termed the RPAP3/R2TP/prefoldin-like complex. Here, we review the literature on the subunits of this complex, which points to a role in nuclear RNAP biogenesis.

Methylation of the DNA/RNA-binding protein Kin17 by METTL22 affects its association with chromatin.

UNLABELLED Kin17 is a protein that was discovered through its immunoreactivity towards an antibody directed against prokaryotic RecA. Further study of Kin17 revealed a function in DNA replication and repair, as well as in pre-mRNA processing. Recently, it was found that Kin17 is methylated on lysine 135 by the newly discovered methyltransferase METTL22. To better understand the function of Kin17 and its regulation by methylation, we used multiple cell compartment protein affinity purification coupled with mass spectrometry (MCC-AP-MS) to identify novel interaction partners of Kin17 and to assess whether these interactions can take place on chromatin. Our results confirm that Kin17 interacts with METTL22 both in the soluble and chromatin fractions. We also show that many RNA-binding proteins, including the previously identified interactor BUD13 as well as spliceosomal and ribosomal subunits, associate with Kin17 in the soluble fraction. Interestingly, overexpression of METTL22 in HEK 293 cells displaces Kin17 from the chromatin to the cytoplasmic fraction, suggesting a role for methylation of lysine 135, a residue that lies within a winged helix domain of Kin17, in regulating association with chromatin. These results are discussed in view of the putative cellular function of Kin17. BIOLOGICAL SIGNIFICANCE The results shown here broaden our understanding of METTL22, a member of a family of newly-discovered non-histone lysine methyltransferases and its substrate, Kin17, a DNA/RNA-binding protein with reported roles in DNA repair and replication and mRNA processing. An innovative method to study protein-protein interactions in multiple cell compartments is employed to outline the interaction network of both proteins. Functional experiments uncover a correlative role between Kin17 lysine methylation and its association with chromatin. This article is part of a Special Issue entitled: Can Proteomics Fill the Gap Between Genomics and Phenotypes?

R2TP/Prefoldin-like component RUVBL1/RUVBL2 directly interacts with ZNHIT2 to regulate assembly of U5 small nuclear ribonucleoprotein

The R2TP/Prefoldin-like (R2TP/PFDL) complex has emerged as a cochaperone complex involved in the assembly of a number of critical protein complexes including snoRNPs, nuclear RNA polymerases and PIKK-containing complexes. Here we report on the use of multiple target affinity purification coupled to mass spectrometry to identify two additional complexes that interact with R2TP/PFDL: the TSC1–TSC2 complex and the U5 small nuclear ribonucleoprotein (snRNP). The interaction between R2TP/PFDL and the U5 snRNP is mostly mediated by the previously uncharacterized factor ZNHIT2. A more general function for the zinc-finger HIT domain in binding RUVBL2 is exposed. Disruption of ZNHIT2 and RUVBL2 expression impacts the protein composition of the U5 snRNP suggesting a function for these proteins in promoting the assembly of the ribonucleoprotein. A possible implication of R2TP/PFDL as a major effector of stress-, energy- and nutrient-sensing pathways that regulate anabolic processes through the regulation of its chaperoning activity is discussed.