ScRpb4, Encoding an RNA Polymerase Subunit from Sugarcane, Is Ubiquitously Expressed and Resilient to Changes in Response to Stress Conditions

RNA polymerase II is an essential multiprotein complex that transcribes thousands of genes, being a fundamental component of the transcription initiation complex. In eukaryotes, RNA polymerase II is formed by a 10-multisubunit conserved core complex, and two additional peripheral subunits, Rpb4 and Rpb7, form the Rpb4/7 subcomplex. Although transcription is vital for cell and organismal viability, little is known about the transcription initiation complex in sugarcane. An initial characterization of the sugarcane RNA polymerase subunit IV (ScRpb4) was performed. Our results demonstrate that ScRpb4 is evolutionarily conserved across kingdoms. At the molecular level, ScRpb4 expression was found in vegetative and reproductive tissues. Furthermore, the expression of ScRpb4 remained stable under various stress conditions, most likely to ensure a proper transcriptional response. Optimal conditions to express ScRpb4 in vitro for further studies were also identified. In this study, an initial characterization of the sugarcane polymerase II subunit IV is presented. Our results open the window to more specific experiments to study ScRpb4 function, for instance, crystal structure determination and pull-down assays as well as their function under biotic and abiotic stresses.

Global proteome response of human cancer cell lines to low dose eIF4E/eIF4G inhibition

<p>Cachexia is a debilitating muscle wasting disease and co-morbidity strongly associated with chronic inflammatory conditions such as cancer, chronic heart failure, chronic obstructive pulmonary disease and sepsis. Cachexia has a strong negative impact on quality of life and research suggests that 20% of cancer patients will die of cachexia. Translation initiation is the most highly regulated step of protein synthesis and the eukaryotic initiation factor 4F (eIF4F) translation initiation complex is the gatekeeper of this process; the eIF4F complex is composed of eIFG, a scaffolding protein, eIF4E, an mRNA cap-recognition protein and eIF4A, an RNA helicase. Inhibition of eIF4A by pateamine A has been shown to rescue muscle wasting in vitro and in vivo, this result has been reproduced with other eIF4A inhibitors. Pateamine A is a sponge-derived natural product with nanomolar toxicity to cancer cells. Surprisingly, at doses well below its anti-neoplastic activity it exerts distinct effects on cachexia. The research in this thesis follows on from previous work in our laboratory with pateamine A in human cell lines. Work on the effects of pateamine A on the proteome suggests that not all the proteins changing in expression are explainable by stressing the translation initiation complex. A model by which motifs in the 5’ UTRs of transcripts are a recognised and removed from the system in a selective manner could help explain these effects. We aimed to target eIF4E, another component of the eIF4F system, with two compounds to see if a comparable dose of eIF4E inhibitors could elicit a pateamine-like response. DMSO, a solvent used extensively in this thesis, had unexpected effects on translation. We conclude that 4E1RCat, a compound developed as a selective inhibitor of eIF4E, is not likely to be useable in further work, due to its window of activity coinciding with an unacceptable concentration of DMSO. Ribavirin, our second compound, showed a proteomic response consistent with its classification as an eIF4E translation initiation inhibitor. The proteome response seen with our eIF4E inhibitors is consistent with disruption of translation initiation. However, the data for 4E1RCat was deemed untrustworthy in the wake of revelations that DMSO, the vehicle in which it is dissolved, exerts an almost identical response. From the results obtained, it was not possible to confidently test whether protein downregulation occurred in response to a 5’UTR sequence motif, as seen for inhibitors of eIF4A. Coupled with the uncertainty associated with the 4E1Rcat results, there were relatively few downregulated proteins from the treatments, and many of these could be explained by the direct biological response to the function of the compound in the treatment. All in all, we have obtained new insights into the effects of DMSO on the proteome which will aid further experimentation. This thesis has laid the groundwork for further investigation of the effects of eIF4F inhibition in the context of better understanding the remediation of cachexia through the eIF4F system.</p>

Global proteome response of human cancer cell lines to low dose eIF4E/eIF4G inhibition

<p>Cachexia is a debilitating muscle wasting disease and co-morbidity strongly associated with chronic inflammatory conditions such as cancer, chronic heart failure, chronic obstructive pulmonary disease and sepsis. Cachexia has a strong negative impact on quality of life and research suggests that 20% of cancer patients will die of cachexia. Translation initiation is the most highly regulated step of protein synthesis and the eukaryotic initiation factor 4F (eIF4F) translation initiation complex is the gatekeeper of this process; the eIF4F complex is composed of eIFG, a scaffolding protein, eIF4E, an mRNA cap-recognition protein and eIF4A, an RNA helicase. Inhibition of eIF4A by pateamine A has been shown to rescue muscle wasting in vitro and in vivo, this result has been reproduced with other eIF4A inhibitors. Pateamine A is a sponge-derived natural product with nanomolar toxicity to cancer cells. Surprisingly, at doses well below its anti-neoplastic activity it exerts distinct effects on cachexia. The research in this thesis follows on from previous work in our laboratory with pateamine A in human cell lines. Work on the effects of pateamine A on the proteome suggests that not all the proteins changing in expression are explainable by stressing the translation initiation complex. A model by which motifs in the 5’ UTRs of transcripts are a recognised and removed from the system in a selective manner could help explain these effects. We aimed to target eIF4E, another component of the eIF4F system, with two compounds to see if a comparable dose of eIF4E inhibitors could elicit a pateamine-like response. DMSO, a solvent used extensively in this thesis, had unexpected effects on translation. We conclude that 4E1RCat, a compound developed as a selective inhibitor of eIF4E, is not likely to be useable in further work, due to its window of activity coinciding with an unacceptable concentration of DMSO. Ribavirin, our second compound, showed a proteomic response consistent with its classification as an eIF4E translation initiation inhibitor. The proteome response seen with our eIF4E inhibitors is consistent with disruption of translation initiation. However, the data for 4E1RCat was deemed untrustworthy in the wake of revelations that DMSO, the vehicle in which it is dissolved, exerts an almost identical response. From the results obtained, it was not possible to confidently test whether protein downregulation occurred in response to a 5’UTR sequence motif, as seen for inhibitors of eIF4A. Coupled with the uncertainty associated with the 4E1Rcat results, there were relatively few downregulated proteins from the treatments, and many of these could be explained by the direct biological response to the function of the compound in the treatment. All in all, we have obtained new insights into the effects of DMSO on the proteome which will aid further experimentation. This thesis has laid the groundwork for further investigation of the effects of eIF4F inhibition in the context of better understanding the remediation of cachexia through the eIF4F system.</p>

Recognition and deubiquitination of free 40S for translational reset by Otu2

In actively translating 80S ribosomes the ribosomal protein eS7 of the 40S subunit is monoubiquitinated by the E3 ligase Not4 and deubiquitinated by the deubiquitination enzyme Otu2 upon ribosomal subunit recycling. Despite its importance for general efficiency of translation the exact role and structural basis for this specific translational reset are only poorly understood. Here we present biochemical and structural data showing that Otu2 can engage the recycled 40S subunit together with the recycling factors ABCE1 and Tma64 immediately after 60S dissociation for mRNA recycling, and that it dissociates before 48S initiation complex formation. A combined structural analysis of Otu2 and Otu2-40S complexes by X-ray crystallography, AlphaFold2 prediction and cryo-EM revealed how Otu2 can specifically be recruited to the 40S, but not to the 80S ribosome, for removal of the eS7-bound ubiquitin moiety. Here, interactions of the largely helical N-terminal domain of Otu2 to sites that are masked and therefore inaccessible in the 80S ribosome are of crucial importance. Collectively, we provide the structural basis for the Otu2 driven deubiquitination step providing a first mechanistic understanding of this translational reset step during ribosome recycling/(re)initiation.

Identification of Enhancers and Promoters in the Genome by Multidimensional Scaling

The positions of enhancers and promoters on genomic DNA remain poorly understood. Chromosomes cannot be observed during the cell division cycle because the genome forms a chromatin structure and spreads within the nucleus. However, high-throughput chromosome conformation capture (Hi-C) measures the physical interactions of genomes. In previous studies, DNA extrusion loops were directly derived from Hi-C heat maps. Multidimensional Scaling (MDS) is used in this assessment to more precisely locate enhancers and promoters. MDS is a multivariate analysis method that reproduces the original coordinates from the distance matrix between elements. We used Hi-C data of cultured osteosarcoma cells and applied MDS as the distance matrix of the genome. In addition, we selected columns 2 and 3 of the orthogonal matrix U as the desired structure. Overall, the DNA loops from the reconstructed genome structure contained bioprocesses involved in transcription, such as the pre-transcriptional initiation complex and RNA polymerase II initiation complex, and transcription factors involved in cancer, such as Foxm1 and CREB3. Therefore, our results are consistent with the biological findings. Our method is suitable for identifying enhancers and promoters in the genome.

Genome-wide regulations of the pre-initiation complex formation and elongating RNA polymerase II by an E3 ubiquitin ligase, San1.

San1 ubiquitin ligase is involved in nuclear protein quality control via its interaction with intrinsically disordered proteins for ubiquitylation and proteasomal degradation. Since several transcription/chromatin regulatory factors contain intrinsically disordered domains and can be inhibitory to transcription when in excess, San1 might be involved in transcription regulation. To address this, we analyzed the role of San1 in genome-wide association of TBP [that nucleates pre-initiation complex (PIC) formation for transcription initiation] and RNA polymerase II (Pol II). Our results reveal the roles of San1 in regulating TBP recruitment to the promoters and Pol II association with the coding sequences, and hence PIC formation and coordination of elongating Pol II, respectively. Consistently, transcription is altered in the absence of San1. Such transcriptional alteration is associated with impaired ubiquitylation and proteasomal degradation of Spt16 and gene association of Paf1, but not the incorporation of centromeric histone, Cse4, into the active genes in Δsan1 . Collectively, our results demonstrate distinct functions of a nuclear protein quality control factor in regulating the genome-wide PIC formation and elongating Pol II (and hence transcription), thus unraveling new gene regulatory mechanisms.

uS5/Rps2 residues at the 40S ribosome entry channel enhance initiation at suboptimal start codons in vivo

The eukaryotic 43S pre-initiation complex (PIC) containing Met-tRNAiMet in a ternary complex (TC) with eIF2-GTP scans the mRNA leader for an AUG codon in favorable “Kozak” context. AUG recognition triggers rearrangement of the PIC from an open conformation to a closed state with more tightly bound Met-tRNAiMet. Yeast ribosomal protein uS5/Rps2 is located at the mRNA entry channel of the 40S subunit in the vicinity of mRNA nucleotides downstream from the AUG codon or rRNA residues that communicate with the decoding center, but its participation in start codon recognition was unknown. We found that non-lethal substitutions of conserved Rps2 residues in the entry channel reduce bulk translation initiation and increase discrimination against poor initiation codons. A subset of these substitutions suppress initiation at near-cognate UUG start codons in a yeast mutant with elevated UUG initiation, and also increase discrimination against AUG codons in suboptimal Kozak context, thus resembling previously described substitutions in uS3/Rps3 at the 40S entry channel or initiation factors eIF1 and eIF1A. In contrast, other Rps2 substitutions selectively discriminate against either near-cognate UUG codons, or poor Kozak context of an AUG or UUG start codon. These findings suggest that different Rps2 residues are involved in distinct mechanisms involved in discriminating against different features of poor initiation sites in vivo.

Requirements for mammalian promoters to decode transcription factor dynamics

In response to different stimuli many transcription factors (TFs) display different activation dynamics that trigger the expression of specific sets of target genes, suggesting that promoters have a way to decode them. Combining optogenetics, deep learning-based image analysis and mathematical modeling, we find that decoding of TF dynamics occurs only when the coupling between TF binding and transcription pre-initiation complex formation is inefficient and that the ability of a promoter to decode TF dynamics gets amplified by inefficient translation initiation. Furthermore, we propose a theoretical mechanism based on phase separation that would allow a promoter to be activated better by pulsatile than sustained TF signals. These results provide an understanding on how TF dynamics are decoded in mammalian cells, which is important to develop optimal strategies to counteract disease conditions, and suggest ways to achieve multiplexing in synthetic pathways.

The role of DDK and Treslin–MTBP in coordinating replication licensing and pre-initiation complex formation

Treslin/Ticrr is required for the initiation of DNA replication and binds to MTBP (Mdm2 Binding Protein). Here, we show that in Xenopus egg extract, MTBP forms an elongated tetramer with Treslin containing two molecules of each protein. Immunodepletion and add-back experiments show that Treslin–MTBP is rate limiting for replication initiation. It is recruited onto chromatin before S phase starts and recruitment continues during S phase. We show that DDK activity both increases and strengthens the interaction of Treslin–MTBP with licensed chromatin. We also show that DDK activity cooperates with CDK activity to drive the interaction of Treslin–MTBP with TopBP1 which is a regulated crucial step in pre-initiation complex formation. These results suggest how DDK works together with CDKs to regulate Treslin–MTBP and plays a crucial in selecting which origins will undergo initiation.