CDK7 kinase activity promotes RNA polymerase II promoter escape by facilitating initiation factor release.

Cyclin-dependent kinase 7 (CDK7), part of the general transcription factor TFIIH, promotes gene transcription by phosphorylating the C-terminal domain of RNA polymerase II (RNA Pol II). Here, we combine rapid CDK7 kinase inhibition with multi-omics analysis to unravel the direct functions of CDK7 in human cells. CDK7 inhibition causes RNA Pol II retention at promoters, leading to decreased RNA Pol II initiation and immediate global downregulation of transcript synthesis. Elongation, termination, and recruitment of co-transcriptional factors are not directly affected. Although RNA Pol II, initiation factors, and Mediator accumulate at promoters, RNA Pol II complexes can also proceed into gene bodies without promoter-proximal pausing while retaining initiation factors and Mediator. Further downstream, RNA Pol II phosphorylation increases and initiation factors and Mediator are released, allowing recruitment of elongation factors and an increase in RNA Pol II elongation velocity. Collectively, CDK7 kinase activity promotes the release of initiation factors and Mediator from RNA Pol II, facilitating RNA Pol II escape from the promoter.

Structural basis of archaeal RNA polymerase transcription elongation and Spt4/5 recruitment.

Archaeal transcription is carried out by a multi-subunit RNA polymerase (RNAP) that is highly homologous in structure and function to eukaryotic RNAP II. Among the set of basal transcription factors, only Spt5 is found in all domains of life, but Spt5 has been shaped during evolution, which is also reflected in the heterodimerization of Spt5 with Spt4 in Archaea and Eukaryotes. To unravel the mechanistic basis of Spt4/5 function in Archaea, we performed structure-function analyses using the archaeal transcriptional machinery of Pyrococcus furiosus (Pfu). We report single-particle cryo-electron microscopy reconstructions of apo RNAP and the archaeal elongation complex (EC) in the absence and presence of Spt4/5. Surprisingly, Pfu Spt4/5 also binds the RNAP in the absence of nucleic acids in a distinct super-contracted conformation. We show that the RNAP clamp/stalk module exhibits conformational flexibility in the apo state of RNAP and that the enzyme contracts upon EC formation or Spt4/5 engagement. We furthermore identified a contact of the Spt5-NGN domain with the DNA duplex that stabilizes the upstream boundary of the transcription bubble and impacts Spt4/5 activity in vitro. This study, therefore, provides the structural basis for Spt4/5 function in archaeal transcription and reveals a potential role beyond the well-described support of elongation.

Structural insights into the cross-exon to cross-intron spliceosome switch.

Early spliceosome assembly can occur through an intron-defined pathway, whereby U1 and U2 small nuclear ribonucleoprotein particles (snRNPs) assemble across the intron1. Alternatively, it can occur through an exon-defined pathway2-5, whereby U2 binds the branch site located upstream of the defined exon and U1 snRNP interacts with the 5' splice site located directly downstream of it. The U4/U6.U5 tri-snRNP subsequently binds to produce a cross-intron (CI) or cross-exon (CE) pre-B complex, which is then converted to the spliceosomal B complex6,7. Exon definition promotes the splicing of upstream introns2,8,9 and plays a key part in alternative splicing regulation10-16. However, the three-dimensional structure of exon-defined spliceosomal complexes and the molecular mechanism of the conversion from a CE-organized to a CI-organized spliceosome, a pre-requisite for splicing catalysis, remain poorly understood. Here cryo-electron microscopy analyses of human CE pre-B complex and B-like complexes reveal extensive structural similarities with their CI counterparts. The results indicate that the CE and CI spliceosome assembly pathways converge already at the pre-B stage. Add-back experiments using purified CE pre-B complexes, coupled with cryo-electron microscopy, elucidate the order of the extensive remodelling events that accompany the formation of B complexes and B-like complexes. The molecular triggers and roles of B-specific proteins in these rearrangements are also identified. We show that CE pre-B complexes can productively bind in trans to a U1 snRNP-bound 5' splice site. Together, our studies provide new mechanistic insights into the CE to CI switch during spliceosome assembly and its effect on pre-mRNA splice site pairing at this stage.

PRPF8-mediated dysregulation of hBrr2 helicase disrupts human spliceosome kinetics and 5´-splice-site selection causing tissue-specific defects.

The carboxy-terminus of the spliceosomal protein PRPF8, which regulates the RNA helicase Brr2, is a hotspot for mutations causing retinitis pigmentosa-type 13, with unclear role in human splicing and tissue-specificity mechanism. We used patient induced pluripotent stem cells-derived cells, carrying the heterozygous PRPF8 c.6926 A > C (p.H2309P) mutation to demonstrate retinal-specific endophenotypes comprising photoreceptor loss, apical-basal polarity and ciliary defects. Comprehensive molecular, transcriptomic, and proteomic analyses revealed a role of the PRPF8/Brr2 regulation in 5'-splice site (5'SS) selection by spliceosomes, for which disruption impaired alternative splicing and weak/suboptimal 5'SS selection, and enhanced cryptic splicing, predominantly in ciliary and retinal-specific transcripts. Altered splicing efficiency, nuclear speckles organisation, and PRPF8 interaction with U6 snRNA, caused accumulation of active spliceosomes and poly(A)+ mRNAs in unique splicing clusters located at the nuclear periphery of photoreceptors. Collectively these elucidate the role of PRPF8/Brr2 regulatory mechanisms in splicing and the molecular basis of retinal disease, informing therapeutic approaches.

Intensity and retention time prediction improves the rescoring of protein-nucleic acid cross-links.

In protein-RNA cross-linking mass spectrometry, UV or chemical cross-linking introduces stable bonds between amino acids and nucleic acids in protein-RNA complexes that are then analyzed and detected in mass spectra. This analytical tool delivers valuable information about RNA-protein interactions and RNA docking sites in proteins, both in vitro and in vivo. The identification of cross-linked peptides with oligonucleotides of different length leads to a combinatorial increase in search space. We demonstrate that the peptide retention time prediction tasks can be transferred to the task of cross-linked peptide retention time prediction using a simple amino acid composition encoding, yielding improved identification rates when the prediction error is included in rescoring. For the more challenging task of including fragment intensity prediction of cross-linked peptides in the rescoring, we obtain, on average, a similar improvement. Further improvement in the encoding and fine-tuning of retention time and intensity prediction models might lead to further gains, and merit further research.

Functional genomics identifies N-acetyllactosamine extension of complex N-glycans as a mechanism to evade lysis by natural killer cells.

Natural killer (NK) cells are primary defenders against cancer precursors, but cancer cells can persist by evading immune surveillance. To investigate the genetic mechanisms underlying this evasion, we perform a genome-wide CRISPR screen using B lymphoblastoid cells. SPPL3, a peptidase that cleaves glycosyltransferases in the Golgi, emerges as a top hit facilitating evasion from NK cytotoxicity. SPPL3-deleted cells accumulate glycosyltransferases and complex N-glycans, disrupting not only binding of ligands to NK receptors but also binding of rituximab, a CD20 antibody approved for treating B cell cancers. Notably, inhibiting N-glycan maturation restores receptor binding and sensitivity to NK cells. A secondary CRISPR screen in SPPL3-deficient cells identifies B3GNT2, a transferase-mediating poly-LacNAc extension, as crucial for resistance. Mass spectrometry confirms enrichment of N-glycans bearing poly-LacNAc upon SPPL3 loss. Collectively, our study shows the essential role of SPPL3 and poly-LacNAc in cancer immune evasion, suggesting a promising target for cancer treatment.

Phosphorylation of ELYS promotes its interaction with VAPB at decondensing chromosomes during mitosis.

ELYS is a nucleoporin that localizes to the nuclear side of the nuclear pore complex (NPC) in interphase cells. In mitosis, it serves as an assembly platform that interacts with chromatin and then with nucleoporin subcomplexes to initiate post-mitotic NPC assembly. Here we identify ELYS as a major binding partner of the membrane protein VAPB during mitosis. In mitosis, ELYS becomes phosphorylated at many sites, including a predicted FFAT (two phenylalanines in an acidic tract) motif, which mediates interaction with the MSP (major sperm protein)-domain of VAPB. Binding assays using recombinant proteins or cell lysates and co-immunoprecipitation experiments show that VAPB binds the FFAT motif of ELYS in a phosphorylation-dependent manner. In anaphase, the two proteins co-localize to the non-core region of the newly forming nuclear envelope. Depletion of VAPB results in prolonged mitosis, slow progression from meta- to anaphase and in chromosome segregation defects. Together, our results suggest a role of VAPB in mitosis upon recruitment to or release from ELYS at the non-core region of the chromatin in a phosphorylation-dependent manner.

Structural basis of Integrator-dependent RNA polymerase II termination.

The Integrator complex can terminate RNA polymerase II (Pol II) in the promoter-proximal region of genes. Previous work has shed light on how Integrator binds to the paused elongation complex consisting of Pol II, the DRB sensitivity-inducing factor (DSIF) and the negative elongation factor (NELF) and how it cleaves the nascent RNA transcript1, but has not explained how Integrator removes Pol II from the DNA template. Here we present three cryo-electron microscopy structures of the complete Integrator-PP2A complex in different functional states. The structure of the pre-termination complex reveals a previously unresolved, scorpion-tail-shaped INTS10-INTS13-INTS14-INTS15 module that may use its 'sting' to open the DSIF DNA clamp and facilitate termination. The structure of the post-termination complex shows that the previously unresolved subunit INTS3 and associated sensor of single-stranded DNA complex (SOSS) factors prevent Pol II rebinding to Integrator after termination. The structure of the free Integrator-PP2A complex in an inactive closed conformation2 reveals that INTS6 blocks the PP2A phosphatase active site. These results lead to a model for how Integrator terminates Pol II transcription in three steps that involve major rearrangements.

Molecular Determinants of Sensitivity to Polatuzumab Vedotin in Diffuse Large B Cell Lymphoma.

Polatuzumab Vedotin (Pola-V) is an antibody-drug conjugate directed to the CD79B subunit of the B cell receptor (BCR). When combined with conventional immunochemotherapy, Pola-V improves outcomes in DLBCL. To identify determinants of Pola-V sensitivity, we used CRISPR-Cas9 screening for genes that modulated Pola-V toxicity for lymphomas or the surface expression of its target, CD79B. Our results reveal the striking impact of CD79B glycosylation on Pola-V epitope availability on the lymphoma cell surface and on Pola-V toxicity. Genetic, pharmacological, and enzymatic approaches that remove sialic acid from N-linked glycans enhanced lymphoma killing by Pola-V. Pola-V toxicity was also modulated by KLHL6, an E3 ubiquitin ligase that is recurrently inactivated in germinal center derived lymphomas. We reveal how KLHL6 targets CD79B for degradation in normal and malignant germinal center B cells, thereby determining expression of the surface BCR complex. Our findings suggest precision medicine strategies to optimize Pola-V as a lymphoma therapeutic.

The thymocyte-specific RNA-binding protein Arpp21 provides TCR repertoire diversity by binding to the 3'-UTR and promoting Rag1 mRNA expression.

The regulation of thymocyte development by RNA-binding proteins (RBPs) is largely unexplored. We identify 642 RBPs in the thymus and focus on Arpp21, which shows selective and dynamic expression in early thymocytes. Arpp21 is downregulated in response to T cell receptor (TCR) and Ca2+ signals. Downregulation requires Stim1/Stim2 and CaMK4 expression and involves Arpp21 protein phosphorylation, polyubiquitination and proteasomal degradation. Arpp21 directly binds RNA through its R3H domain, with a preference for uridine-rich motifs, promoting the expression of target mRNAs. Analysis of the Arpp21-bound transcriptome reveals strong interactions with the Rag1 3'-UTR. Arpp21-deficient thymocytes show reduced Rag1 expression, delayed TCR rearrangement and a less diverse TCR repertoire. This phenotype is recapitulated in Rag1 3'-UTR mutant mice harboring a deletion of the Arpp21 response region. These findings show how thymocyte-specific Arpp21 promotes Rag1 expression to enable TCR repertoire diversity until signals from the TCR terminate Arpp21 and Rag1 activities.

Structural mechanisms of autoinhibition and substrate recognition by the ubiquitin ligase HACE1.

Ubiquitin ligases (E3s) are pivotal specificity determinants in the ubiquitin system by selecting substrates and decorating them with distinct ubiquitin signals. However, structure determination of the underlying, specific E3-substrate complexes has proven challenging owing to their transient nature. In particular, it is incompletely understood how members of the catalytic cysteine-driven class of HECT-type ligases (HECTs) position substrate proteins for modification. Here, we report a cryogenic electron microscopy (cryo-EM) structure of the full-length human HECT HACE1, along with solution-based conformational analyses by small-angle X-ray scattering and hydrogen-deuterium exchange mass spectrometry. Structure-based functional analyses in vitro and in cells reveal that the activity of HACE1 is stringently regulated by dimerization-induced autoinhibition. The inhibition occurs at the first step of the catalytic cycle and is thus substrate-independent. We use mechanism-based chemical crosslinking to reconstitute a complex of activated, monomeric HACE1 with its major substrate, RAC1, determine its structure by cryo-EM and validate the binding mode by solution-based analyses. Our findings explain how HACE1 achieves selectivity in ubiquitinating the active, GTP-loaded state of RAC1 and establish a framework for interpreting mutational alterations of the HACE1-RAC1 interplay in disease. More broadly, this work illuminates central unexplored aspects in the architecture, conformational dynamics, regulation and specificity of full-length HECTs.

Structural basis for RNA polymerase II ubiquitylation and inactivation in transcription-coupled repair.

During transcription-coupled DNA repair (TCR), RNA polymerase II (Pol II) transitions from a transcriptionally active state to an arrested state that allows for removal of DNA lesions. This transition requires site-specific ubiquitylation of Pol II by the CRL4CSA ubiquitin ligase, a process that is facilitated by ELOF1 in an unknown way. Using cryogenic electron microscopy, biochemical assays and cell biology approaches, we found that ELOF1 serves as an adaptor to stably position UVSSA and CRL4CSA on arrested Pol II, leading to ligase neddylation and activation of Pol II ubiquitylation. In the presence of ELOF1, a transcription factor IIS (TFIIS)-like element in UVSSA gets ordered and extends through the Pol II pore, thus preventing reactivation of Pol II by TFIIS. Our results provide the structural basis for Pol II ubiquitylation and inactivation in TCR.

Cryo-EM analyses of dimerized spliceosomes provide new insights into the functions of B complex proteins.

The B complex is a key intermediate stage of spliceosome assembly. To improve the structural resolution of monomeric, human spliceosomal B (hB) complexes and thereby generate a more comprehensive hB molecular model, we determined the cryo-EM structure of B complex dimers formed in the presence of ATP γ S. The enhanced resolution of these complexes allows a finer molecular dissection of how the 5' splice site (5'ss) is recognized in hB, and new insights into molecular interactions of FBP21, SNU23 and PRP38 with the U6/5'ss helix and with each other. It also reveals that SMU1 and RED are present as a heterotetrameric complex and are located at the interface of the B dimer protomers. We further show that MFAP1 and UBL5 form a 5' exon binding channel in hB, and elucidate the molecular contacts stabilizing the 5' exon at this stage. Our studies thus yield more accurate models of protein and RNA components of hB complexes. They further allow the localization of additional proteins and protein domains (such as SF3B6, BUD31 and TCERG1) whose position was not previously known, thereby uncovering new functions for B-specific and other hB proteins during pre-mRNA splicing.

RNA helicase IGHMBP2 regulates THO complex to ensure cellular mRNA homeostasis.

RNA helicases constitute a large protein family implicated in cellular RNA homeostasis and disease development. Here, we show that the RNA helicase IGHMBP2, linked to the neuromuscular disorder spinal muscular atrophy with respiratory distress type 1 (SMARD1), associates with polysomes and impacts translation of mRNAs containing short, GC-rich, and structured 5' UTRs. The absence of IGHMBP2 causes ribosome stalling at the start codon of target mRNAs, leading to reduced translation efficiency. The main mRNA targets of IGHMBP2-mediated regulation encode for components of the THO complex (THOC), linking IGHMBP2 to mRNA production and nuclear export. Accordingly, failure of IGHMBP2 regulation of THOC causes perturbations of the transcriptome and its encoded proteome, and ablation of THOC subunits phenocopies these changes. Thus, IGHMBP2 is an upstream regulator of THOC. Of note, IGHMBP2-dependent regulation of THOC is also observed in astrocytes derived from patients with SMARD1 disease, suggesting that deregulated mRNA metabolism contributes to SMARD1 etiology and may enable alternative therapeutic avenues.

Protein degradation by human 20S proteasomes elucidates the interplay between peptide hydrolysis and splicing.

If and how proteasomes catalyze not only peptide hydrolysis but also peptide splicing is an open question that has divided the scientific community. The debate has so far been based on immunopeptidomics, in vitro digestions of synthetic polypeptides as well as ex vivo and in vivo experiments, which could only indirectly describe proteasome-catalyzed peptide splicing of full-length proteins. Here we develop a workflow-and cognate software - to analyze proteasome-generated non-spliced and spliced peptides produced from entire proteins and apply it to in vitro digestions of 15 proteins, including well-known intrinsically disordered proteins such as human tau and α-Synuclein. The results confirm that 20S proteasomes produce a sizeable variety of cis-spliced peptides, whereas trans-spliced peptides are a minority. Both peptide hydrolysis and splicing produce peptides with well-defined characteristics, which hint toward an intricate regulation of both catalytic activities. At protein level, both non-spliced and spliced peptides are not randomly localized within protein sequences, but rather concentrated in hotspots of peptide products, in part driven by protein sequence motifs and proteasomal preferences. At sequence level, the different peptide sequence preference of peptide hydrolysis and peptide splicing suggests a competition between the two catalytic activities of 20S proteasomes during protein degradation.

Response to Bruton's tyrosine kinase inhibitors in aggressive lymphomas linked to chronic selective autophagy.

Diffuse large B cell lymphoma (DLBCL) is an aggressive, profoundly heterogeneous cancer, presenting a challenge for precision medicine. Bruton's tyrosine kinase (BTK) inhibitors block B cell receptor (BCR) signaling and are particularly effective in certain molecular subtypes of DLBCL that rely on chronic active BCR signaling to promote oncogenic NF-κB. The MCD genetic subtype, which often acquires mutations in the BCR subunit, CD79B, and in the innate immune adapter, MYD88L265P, typically resists chemotherapy but responds exceptionally to BTK inhibitors. However, the underlying mechanisms of response to BTK inhibitors are poorly understood. Herein, we find a non-canonical form of chronic selective autophagy in MCD DLBCL that targets ubiquitinated MYD88L265P for degradation in a TBK1-dependent manner. MCD tumors acquire genetic and epigenetic alterations that attenuate this autophagic tumor suppressive pathway. In contrast, BTK inhibitors promote autophagic degradation of MYD88L265P, thus explaining their exceptional clinical benefit in MCD DLBCL.

Phosphatases modified by LH signaling in ovarian follicles: testing their role in regulating the NPR2 guanylyl cyclase†.

In response to luteinizing hormone (LH), multiple proteins in rat and mouse granulosa cells are rapidly dephosphorylated, but the responsible phosphatases remain to be identified. Because the phosphorylation state of phosphatases can regulate their interaction with substrates, we searched for phosphatases that might function in LH signaling by using quantitative mass spectrometry. We identified all proteins in rat ovarian follicles whose phosphorylation state changed detectably in response to a 30-min exposure to LH, and within this list, identified protein phosphatases or phosphatase regulatory subunits that showed changes in phosphorylation. Phosphatases in the phosphoprotein phosphatase (PPP) family were of particular interest because of their requirement for dephosphorylating the natriuretic peptide receptor 2 (NPR2) guanylyl cyclase in the granulosa cells, which triggers oocyte meiotic resumption. Among the PPP family regulatory subunits, PPP1R12A and PPP2R5D showed the largest increases in phosphorylation, with 4-10 fold increases in signal intensity on several sites. Although follicles from mice in which these phosphorylations were prevented by serine-to-alanine mutations in either Ppp1r12a or Ppp2r5d showed normal LH-induced NPR2 dephosphorylation, these regulatory subunits and others could act redundantly to dephosphorylate NPR2. Our identification of phosphatases and other proteins whose phosphorylation state is rapidly modified by LH provides clues about multiple signaling pathways in ovarian follicles.

The previously uncharacterized RnpM (YlxR) protein modulates the activity of ribonuclease P in Bacillus subtilis in vitro.

Even though Bacillus subtilis is one of the most studied organisms, no function has been identified for about 20% of its proteins. Among these unknown proteins are several RNA- and ribosome-binding proteins suggesting that they exert functions in cellular information processing. In this work, we have investigated the RNA-binding protein YlxR. This protein is widely conserved in bacteria and strongly constitutively expressed in B. subtilis suggesting an important function. We have identified the RNA subunit of the essential RNase P as the binding partner of YlxR. The main activity of RNase P is the processing of 5' ends of pre-tRNAs. In vitro processing assays demonstrated that the presence of YlxR results in reduced RNase P activity. Chemical cross-linking studies followed by in silico docking analysis and experiments with site-directed mutant proteins suggest that YlxR binds to the region of the RNase P RNA that is important for binding and cleavage of the pre-tRNA substrate. We conclude that the YlxR protein is a novel interaction partner of the RNA subunit of RNase P that serves to finetune RNase P activity to ensure appropriate amounts of mature tRNAs for translation. We rename the YlxR protein RnpM for RNase P modulator.

Nedd4-2-dependent regulation of astrocytic Kir4.1 and Connexin43 controls neuronal network activity.

Nedd4-2 is an E3 ubiquitin ligase in which missense mutation is related to familial epilepsy, indicating its critical role in regulating neuronal network activity. However, Nedd4-2 substrates involved in neuronal network function have yet to be identified. Using mouse lines lacking Nedd4-1 and Nedd4-2, we identified astrocytic channel proteins inwardly rectifying K+ channel 4.1 (Kir4.1) and Connexin43 as Nedd4-2 substrates. We found that the expression of Kir4.1 and Connexin43 is increased upon conditional deletion of Nedd4-2 in astrocytes, leading to an elevation of astrocytic membrane ion permeability and gap junction activity, with a consequent reduction of γ-oscillatory neuronal network activity. Interestingly, our biochemical data demonstrate that missense mutations found in familial epileptic patients produce gain-of-function of the Nedd4-2 gene product. Our data reveal a process of coordinated astrocytic ion channel proteostasis that controls astrocyte function and astrocyte-dependent neuronal network activity and elucidate a potential mechanism by which aberrant Nedd4-2 function leads to epilepsy.

GPATCH4 regulates rRNA and snRNA 2'-O-methylation in both DHX15-dependent and DHX15-independent manners.

Regulation of RNA helicase activity, often accomplished by protein cofactors, is essential to ensure target specificity within the complex cellular environment. The largest family of RNA helicase cofactors are the G-patch proteins, but the cognate RNA helicases and cellular functions of numerous human G-patch proteins remain elusive. Here, we discover that GPATCH4 is a stimulatory cofactor of DHX15 that interacts with the DEAH box helicase in the nucleolus via residues in its G-patch domain. We reveal that GPATCH4 associates with pre-ribosomal particles, and crosslinks to the transcribed ribosomal DNA locus and precursor ribosomal RNAs as well as binding to small nucleolar- and small Cajal body-associated RNAs that guide rRNA and snRNA modifications. Loss of GPATCH4 impairs 2'-O-methylation at various rRNA and snRNA sites leading to decreased protein synthesis and cell growth. We demonstrate that the regulation of 2'-O-methylation by GPATCH4 is both dependent on, and independent of, its interaction with DHX15. Intriguingly, the ATPase activity of DHX15 is necessary for efficient methylation of DHX15-dependent sites, suggesting a function of DHX15 in regulating snoRNA-guided 2'-O-methylation of rRNA that requires activation by GPATCH4. Overall, our findings extend knowledge on RNA helicase regulation by G-patch proteins and also provide important new insights into the mechanisms regulating installation of rRNA and snRNA modifications, which are essential for ribosome function and pre-mRNA splicing.