Co-transcriptional Processing Research Articles

Recent Insights into Plant miRNA Biogenesis: Multiple Layers of miRNA Level Regulation.

MicroRNAs are small RNAs, 20-22 nt long, the main role of which is to downregulate gene expression at the level of mRNAs. MiRNAs are fundamental regulators of plant growth and development in response to internal signals as well as in response to abiotic and biotic factors. Therefore, the deficiency or excess of individual miRNAs is detrimental to particular aspects of a plant's life. In consequence, the miRNA levels must be appropriately adjusted. To obtain proper expression of each miRNA, their biogenesis is controlled at multiple regulatory layers. Here, we addressed processes discovered to influence miRNA steady-state levels, such as MIR transcription, co-transcriptional pri-miRNA processing (including splicing, polyadenylation, microprocessor assembly and activity) and miRNA-encoded peptides synthesis. MiRNA stability, RISC formation and miRNA export out of the nucleus and out of the plant cell also define the levels of miRNAs in various plant tissues. Moreover, we show the evolutionary conservation of miRNA biogenesis core proteins across the plant kingdom.

Mechanisms of circular RNA degradation.

Circular RNAs (CircRNAs) are a class of noncoding RNAs formed by backsplicing during cotranscriptional and posttranscriptional processes, and they widely exist in various organisms. CircRNAs have multiple biological functions and are associated with the occurrence and development of many diseases. While the biogenesis and biological function of circRNAs have been extensively studied, there are few studies on circRNA degradation and only a few pathways for specific circRNA degradation have been identified. Here we outline basic information about circRNAs, summarize the research on the circRNA degradation mechanisms and discusses where this field might head, hoping to provide some inspiration and guidance for scholars who aim to study the degradation of circRNAs.

Rate of transcription elongation and sequence-specific pausing by RNA polymerase I directly influence rRNA processing

One of the first steps in ribosome biogenesis is transcription of the ribosomal DNA by RNA polymerase I (Pol I). Processing of the resultant rRNA begins cotranscriptionally, and perturbation of Pol I transcription elongation results in defective rRNA processing. Mechanistic insight regarding the link between transcription elongation and ribosome assembly is lacking because of limited invivo methods to assay Pol I transcription. Here, we use native elongating transcript sequencing (NET-Seq) with a strain of Saccharomyces cerevisiae containing a point mutation in Pol I, rpa190-F1205H, which results in impaired rRNA processing and ribosome assembly. We previously demonstrated that this mutation caused a mild reduction in the transcription elongation rate of Pol I invitro; however, transcription elongation by the mutant has not been characterized invivo. Here, our findings demonstrate that the mutant Pol I has an increased pause propensity during processive transcription elongation both invitro and invivo. NET-Seq reveals that rpa190-F1205H Pol I displays alternative pause site preferences invivo. Specifically, the mutant is sensitized to A/G residues in the RNA:DNA hybrid and at the last incorporated nucleotide position. Furthermore, both NET-Seq and EM analysis of Miller chromatin spreads reveal pileups of rpa190-F1205H Pol I throughout the ribosomal DNA, particularly at the 5' end of the 35S gene. This combination of invitro and invivo analyses of a Pol I mutant provides novel insights into Pol I elongation properties and indicates how these properties are crucial for efficient cotranscriptional rRNA processing and ribosome assembly.

Chromatin-associated microprocessor assembly is regulated by the U1 snRNP auxiliary protein PRP40.

In plants, microRNA (miRNA) biogenesis involves cotranscriptional processing of RNA polymerase II (RNAPII)-generated primary transcripts by a multi-protein complex termed the microprocessor. Here, we report that Arabidopsis (Arabidopsis thaliana) PRE-MRNA PROCESSING PROTEIN 40 (PRP40), the U1 snRNP auxiliary protein, positively regulates the recruitment of SERRATE, a core component of the plant microprocessor, to miRNA genes. The association of DICER-LIKE1 (DCL1), the microprocessor endoribonuclease, with chromatin was altered in prp40ab mutant plants. Impaired cotranscriptional microprocessor assembly was accompanied by RNAPII accumulation at miRNA genes and retention of miRNA precursors at their transcription sites in the prp40ab mutant plants. We show that cotranscriptional microprocessor assembly, regulated by AtPRP40, positively affects RNAPII transcription of miRNA genes and is important to reach the correct levels of produced miRNAs.

Molecular dynamics simulation on the Thermosinus carboxydivorans pfl ZTP riboswitch by ligand binding

Riboswitches are RNA molecules that can regulate gene expression which is affected by ligand-binding during cotranscriptional folding process. However, the role of ligand during the folding is still unclear. In this study, the pfl domain of Thermosinus carboxydivorans ZTP riboswitch was discussed. The ligand is molecule ZMP. We mainly analyzed the change of ZMP-free and ZMP-bound aptamer domain by the dynamics simulation method. Structural features by calculating their RMSD, RMSF, etc. are analyzed. The results demonstrate that the binding domain require the presence of ZMP to maintain a stable fold. It also suggested that ZMP specificly binding to ZTP can generate more hydrogen bonds in the binding domain. Through the calculation of binding free energy decomposition of each nucleotide, molecule ZMP was found to promote the recognition and binding process of ligands by controlling some special nucleotides in the process of ligand binding. At last, the dynamical correlation and components of conformational motions were both applied to explore the effect of molecule ZMP to ZTP riboswitch. In general, ZMP can effectively affect the motions of the pfl riboswitch and facilitate the folding process of the ZTP riboswitch.These results may provide some new ideas for structural changes in riboswitches and their cotranscriptional folding process.

Widespread hydroxylation of unstructured lysine-rich protein domains by JMJD6

The Jumonji domain-containing protein JMJD6 is a 2-oxoglutarate-dependent dioxygenase associated with a broad range of biological functions. Cellular studies have implicated the enzyme in chromatin biology, transcription, DNA repair, mRNA splicing, and cotranscriptional processing. Although not all studies agree, JMJD6 has been reported to catalyze both hydroxylation of lysine residues and demethylation of arginine residues. However, despite extensive study and indirect evidence for JMJD6 catalysis in many cellular processes, direct assignment of JMJD6 catalytic substrates has been limited. Examination of a reported site of proline hydroxylation within a lysine-rich region of the tandem bromodomain protein BRD4 led us to conclude that hydroxylation was in fact on lysine and catalyzed by JMJD6. This prompted a wider search for JMJD6-catalyzed protein modifications deploying mass spectrometric methods designed to improve the analysis of such lysine-rich regions. Using lysine derivatization with propionic anhydride to improve the analysis of tryptic peptides and nontryptic proteolysis, we report 150 sites of JMJD6-catalyzed lysine hydroxylation on 48 protein substrates, including 19 sites of hydroxylation on BRD4. Most hydroxylations were within lysine-rich regions that are predicted to be unstructured; in some, multiple modifications were observed on adjacent lysine residues. Almost all of the JMJD6 substrates defined in these studies have been associated with membraneless organelle formation. Given the reported roles of lysine-rich regions in subcellular partitioning by liquid-liquid phase separation, our findings raise the possibility that JMJD6 may play a role in regulating such processes in response to stresses, including hypoxia.

Anomalous HIV-1 RNA, How Cap-Methylation Segregates Viral Transcripts by Form and Function.

The acquisition of m7G-cap-binding proteins is now recognized as a major variable driving the form and function of host RNAs. This manuscript compares the 5′-cap-RNA binding proteins that engage HIV-1 precursor RNAs, host mRNAs, small nuclear (sn)- and small nucleolar (sno) RNAs and sort into disparate RNA-fate pathways. Before completion of the transcription cycle, the transcription start site of nascent class II RNAs is appended to a non-templated guanosine that is methylated (m7G-cap) and bound by hetero-dimeric CBP80-CBP20 cap binding complex (CBC). The CBC is a nexus for the co-transcriptional processing of precursor RNAs to mRNAs and the snRNA and snoRNA of spliceosomal and ribosomal ribonucleoproteins (RNPs). Just as sn/sno-RNAs experience hyper-methylation of m7G-cap to trimethylguanosine (TMG)-cap, so do select HIV RNAs and an emerging cohort of mRNAs. TMG-cap is blocked from Watson:Crick base pairing and disqualified from participating in secondary structure. The HIV TMG-cap has been shown to license select viral transcripts for specialized cap-dependent translation initiation without eIF4E that is dependent upon CBP80/NCBP3. The exceptional activity of HIV precursor RNAs secures their access to maturation pathways of sn/snoRNAs, canonical and non-canonical host mRNAs in proper stoichiometry to execute the retroviral replication cycle.

R-loops at microRNA encoding loci promote co-transcriptional processing of pri-miRNAs in plants

In most organisms, the maturation of nascent RNAs is coupled to transcription. Unlike in animals, the RNA polymerase II (RNAPII) transcribes microRNA genes (MIRNAs) as long and structurally variable pri-miRNAs in plants. Current evidence suggests that the miRNA biogenesis complex assembly initiates early during the transcription of pri-miRNAs in plants. However, it is unknown whether miRNA processing occurs co-transcriptionally. Here, we used native elongating transcript sequencing data and imaging techniques to demonstrate that plant miRNA biogenesis occurs coupled to transcription. We found that the entire biogenesis occurs co-transcriptionally for pri-miRNAs processed from the loop of the hairpin but requires a second nucleoplasmic step for those processed from the base. Furthermore, we found that co- and post-transcriptional miRNA processing mechanisms co-exist for most miRNAs in a dynamic balance. Notably, we discovered that R-loops, formed near the transcription start site region of MIRNAs, promote co-transcriptional pri-miRNA processing. Furthermore, our results suggest the neofunctionalization of co-transcriptionally processed miRNAs, boosting countless regulatory scenarios.

ADAR1 Prevents R-loop Accumulation-Driven ATR Pathway Activation in Ovarian Cancer.

Adenosine (A)-to-inosine (I) RNA editing is the most prevalent RNA editing mechanism, in which adenosine deaminase acting on RNA 1 (ADAR1) is a major adenosine deaminase. Increasing evidence suggests that editing dysregulation of ADAR1 plays an important role in human tumorigenesis, while the underlying mechanism remains elusive. Here, we demonstrated that ADAR1 was highly expressed in ovarian cancer tissues and negatively correlated with progression free survival of ovarian cancer patients. Importantly, silence of ADAR1 repressed ovarian cancer cell growth and colony formation in vitro and inhibited ovarian cancer cell tumorigenesis in vivo. Further cell cycle and transcriptome profile analysis revealed that silence of ADAR1 in ovarian cancer cells induced cell cycle arrest at G1/G0 stage. Mechanistically, loss of ADAR1 caused R-loop abnormal accumulation, thereby contributing to single stand DNA break and ATR pathway activation. Additionally, ADAR1 interacted with DHX9 to regulate R-loop complex formation, and A-to-I editing of nascent RNA repressed R-loop formation during co-transcriptional process. Together, our results identify a novel ADAR1/R-loop/ATR axis critical for ovarian cancer progression and a potential target for ovarian cancer therapy.

Antagonistic cotranscriptional regulation through ARGONAUTE1 and the THO/TREX complex orchestrates FLC transcriptional output

Quantitative transcriptional control is essential for physiological and developmental processes in many organisms. Transcriptional output is influenced by cotranscriptional processes interconnected to chromatin regulation, but how the functions of different cotranscriptional regulators are integrated is poorly understood. The Arabidopsis floral repressor locus FLOWERING LOCUS C (FLC) is cotranscriptionally repressed by alternative processing of the antisense transcript COOLAIR. Proximal 3'-end processing of COOLAIR resolves a cotranscriptionally formed R-loop, and this process physically links to a histone-modifying complex FLD/SDG26/LD. This induces a chromatin environment locally that determines low transcription initiation and a slow elongation rate to both sense and antisense strands. Here, we show that ARGONAUTE1 (AGO1) genetically functions in this cotranscriptional repression mechanism. AGO1 associates with COOLAIR and influences COOLAIR splicing dynamics to promote proximal COOLAIR, R-loop resolution, and chromatin silencing. Proteomic analyses revealed physical associations between AGO1, subunits of RNA Polymerase II (Pol II), the splicing-related proteins-the spliceosome NineTeen Complex (NTC) and related proteins (NTR)-and the THO/TREX complex. We connect these activities by demonstrating that the THO/TREX complex activates FLC expression acting antagonistically to AGO1 in COOLAIR processing. Together these data reveal that antagonistic cotranscriptional regulation through AGO1 or THO/TREX influences COOLAIR processing to deliver a local chromatin environment that determines FLC transcriptional output. The involvement of these conserved cotranscriptional regulators suggests similar mechanisms may underpin quantitative transcriptional regulation generally.

Abstract PO-034: CPSF3 inhibition halts pancreatic cancer cell proliferation by limiting core histone supplies

Abstract Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal malignancies with few effective treatment options. Therefore, the need for new therapeutic interventions is urgent. Recently, we have revealed that the gene regulatory process alternative polyadenylation (APA) is highly dysregulated in PDAC patients. APA is a co-transcriptional process that generates distinct transcript isoforms with different 3’ untranslated region (UTR) length, which regulates mRNA stability and gene and protein expression. We found that APA promotes dysregulation of PDAC-promoting genes, predicts poor prognosis in PDAC patients and identified a novel APA-regulated drug target (Venkat et al., Genome Research, 2020). These findings raise the question of whether targeting APA could be a viable therapeutic strategy for PDAC treatment. APA is controlled by a large protein complex assembled on the pre-mRNA, including the cleavage and polyadenylation specificity factor 3 (CPSF3). CPSF3 is an endonuclease that cleaves the pre-mRNA before addition of the polyA tail. Notably, CPSF3 is also a subunit of the histone pre-mRNA processing complex and is critical for the cleavage and processing of core histones. Recently, it was determined that JTE-607, a drug with a long history of safety in humans but no known mechanism of action, is a CPSF3 inhibitor. Subsequently, CPSF3 has been shown to be required for growth of acute myeloid leukemia and Ewing sarcoma cells. However, whether CPSF3 is a potential target in adenocarcinoma is yet to be explored. In PDAC patients, CPSF3 is highly expressed and is associated with unfavorable prognosis. Knockdown or inhibition of CPSF3 decreases PDAC cell proliferation and clonogenicity while having no effect on the growth of non-transformed pancreatic cell lines. Cell cycle analysis and BrdU incorporation assays determined that CPSF3 inhibition arrests cells in early S-phase of the cell cycle. As maintenance of high histone levels are important for the packaging of newly synthesized DNA and progression through the cell cycle, we hypothesized that CPSF3 inhibition decreases PDAC cell proliferation by reducing histone levels. Indeed, CPSF3 inhibition decreased core histone protein levels in a time- and dose-dependent manner. High throughput screening revealed a synergistic effect between JTE-607 and Abexinostat, an HDAC inhibitor, suggesting a novel therapeutic strategy. Therefore, we identify CPSF3 as a druggable target in PDAC and reveal a novel mechanism by which CPSF3 disruption attenuates cell proliferation. Citation Format: Abdulrahman A. Alahmari, Carla Schwarz, Emily Paterson, Swati Venkat, Arwen Tisdale, Michael E. Feigin. CPSF3 inhibition halts pancreatic cancer cell proliferation by limiting core histone supplies [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PO-034.

Analysis of differential gene expression and transcript usage in hippocampus of Apoe null mutant mice: Implications for Alzheimer’s disease

A dataset of single-nucleus RNA sequencing (snRNAseq) data was analyzed using Seurat, Sierra, and Ingenuity Pathway Analysis (IPA) programs to assess differentially expressed genes (DEGs) and differential transcript usage (DTU) in mouse hippocampal cell types. Seurat identified DEGs between the wild type (WT) and Apoe knockout (EKO) mice. IPA identified 11 statistically significant canonical pathways in >1 cell type. Sierra identified Sipa1l1 with DTU between WT and EKO samples. Analysis of the Sipa1l1 peak region identified an alternative non-canonical polyadenylation signal and a putative cytoplasmic polyadenylation element. APOE regulation of gene transcription and co-transcriptional RNA processing may underlie Alzheimer’s disease.

Cotranscriptional folding of RNA pseudoknots with different rates

We investigate and compare the refolding and cotranscriptional folding process of RNA pseudoknot including PK5 pseudoknot, human telomerase RNA (hTR) pseudoknot and its mutants by using helix-based transition rate model and master equation. It is found that in the refolding process, the final states for PKWT and two mutants PK179AG/110CU and PK△U177 are all pseudoknots. However, the final state of mutant PKDC is a stable long helix. Furthermore, the cotranscriptional folding intermediates are quite different from refolding ones. The intermediate found in refolding process for PKWT is also shown during cotranscription. For PK179AG/110CU, the two intermediates in refolding are not found in cotranscriptional folding. For PK△U177, only one of the intermediates found in refolding is not observed in cotranscription. For PKDC, the unfolding sequence in the refolding process directly transits to native helix. Two intermediates are found during cotranscription. The populations of intermediates and native structure also depend on the cotranscriptional folding rate. All these findings can offer useful insights into cotranscription folding of hTR pseudoknot with different rates.

Nuclear compartmentalization as a mechanism of quantitative control of gene expression.

Gene regulation requires the dynamic coordination of hundreds of regulatory factors at precise genomic and RNA targets. Although many regulatory factors have specific affinity for their nucleic acid targets, molecular diffusion and affinity models alone cannot explain many of the quantitative features of gene regulation in the nucleus. One emerging explanation for these quantitative properties is that DNA, RNA and proteins organize within precise, 3D compartments in the nucleus to concentrate groups of functionally related molecules. Recently, nucleic acids and proteins involved in many important nuclear processes have been shown to engage in cooperative interactions, which lead to the formation of condensates that partition the nucleus. In this Review, we discuss an emerging perspective of gene regulation, which moves away from classic models of stoichiometric interactions towards an understanding of how spatial compartmentalization can lead to non-stoichiometric molecular interactions and non-linear regulatory behaviours. We describe key mechanisms of nuclear compartment formation, including emerging roles for non-coding RNAs in facilitating their formation, and discuss the functional role of nuclear compartments in transcription regulation, co-transcriptional and post-transcriptional RNA processing, and higher-order chromatin regulation. More generally, we discuss how compartmentalization may explain important quantitative aspects of gene regulation.

HP1γ binding pre-mRNA intronic repeatsmodulates RNA splicing decisions.

HP1 proteins are best known as markers of heterochromatin and gene silencing. Yet, they are also RNA-binding proteins and the HP1γ/CBX3 family member is present on transcribed genes together with RNA polymerase II, where it regulates co-transcriptional processes such as alternative splicing. To gain insight in the role of the RNA-binding activity of HP1γ in transcriptionally active chromatin, we have captured and analysed RNAs associated with this protein. We find that HP1γ is specifically targeted to hexameric RNA motifs and coincidentally transposable elements of the SINE family. As these elements are abundant in introns, while essentially absent from exons, the HP1γ RNA association tethers unspliced pre-mRNA to chromatin via the intronic regions and limits the usage of intronic cryptic splice sites. Thus, our data unveil novel determinants in the relationship between chromatin and co-transcriptional splicing.

RNA polymerase II speed: a key player in controlling and adapting transcriptome composition.

RNA polymerase II (RNA Pol II) speed or elongation rate, i.e., the number of nucleotides synthesized per unit of time, is a major determinant of transcriptome composition. It controls co‐transcriptional processes such as splicing, polyadenylation, and transcription termination, thus regulating the production of alternative splice variants, circular RNAs, alternatively polyadenylated transcripts, or read‐through transcripts. RNA Pol II speed itself is regulated in response to intra‐ and extra‐cellular stimuli and can in turn affect the transcriptome composition in response to these stimuli. Evidence points to a potentially important role of transcriptome composition modification through RNA Pol II speed regulation for adaptation of cells to a changing environment, thus pointing to a function of RNA Pol II speed regulation in cellular physiology. Analyzing RNA Pol II speed dynamics may therefore be central to fully understand the regulation of physiological processes, such as the development of multicellular organisms. Recent findings also raise the possibility that RNA Pol II speed deregulation can be detrimental and participate in disease progression. Here, we review initial and current approaches to measure RNA Pol II speed, as well as providing an overview of the factors controlling speed and the co‐transcriptional processes which are affected. Finally, we discuss the role of RNA Pol II speed regulation in cell physiology.

Regulation of polyphosphate glucokinase gene expression through cotranscriptional processing in Mycobacterium tuberculosis H37Rv.

Transcription is a molecular process that involves the synthesis of RNA chain into the 5'-3' direction, and simultaneously nascent RNA chain tends to form geometric structures, known as cotranscriptional folding. This folding determines the functional properties of RNA molecules and possibly has a critical role during the synthesis. This functioning includes the characterized properties of riboswitches and ribozymes, which are significant when the transcription rate is comparable to the cellular environment. This study reports a novel noncoding region important in the genetic expression of polyphosphate glucokinase (ppgk) in Mycobacterium tuberculosis. This noncoding element of ppgk gene undergoes cleavage activity during the transcriptional process in M.tuberculosis. We revealed that cleavage occurs within the nascent RNA, and the resultant cleaved 3'RNA fragment carries the Shine-Dalgarno (SD) sequence and expression platform. We concluded cotranscriptional processing at the noncoding region as the required mechanism for ppgk expression that remains constitutive within the bacterial environment. This study defines the molecular mechanism dependent on the transient but highly active structural features of the nascent RNA.

Regulation of Eukaryotic RNAPs Activities by Phosphorylation.

Evolutionarily conserved kinases and phosphatases regulate RNA polymerase II (RNAPII) transcript synthesis by modifying the phosphorylation status of the carboxyl-terminal domain (CTD) of Rpb1, the largest subunit of RNAPII. Proper levels of Rpb1-CTD phosphorylation are required for RNA co-transcriptional processing and to coordinate transcription with other nuclear processes, such as chromatin remodeling and histone modification. Whether other RNAPII subunits are phosphorylated and influences their role in gene expression is still an unanswered question. Much less is known about RNAPI and RNAPIII phosphorylation, whose subunits do not contain functional CTDs. However, diverse studies have reported that several RNAPI and RNAPIII subunits are susceptible to phosphorylation. Some of these phosphorylation sites are distributed within subunits common to all three RNAPs whereas others are only shared between RNAPI and RNAPIII. This suggests that the activities of all RNAPs might be finely modulated by phosphorylation events and raises the idea of a tight coordination between the three RNAPs. Supporting this view, the transcription by all RNAPs is regulated by signaling pathways that sense different environmental cues to adapt a global RNA transcriptional response. This review focuses on how the phosphorylation of RNAPs might regulate their function and we comment on the regulation by phosphorylation of some key transcription factors in the case of RNAPI and RNAPIII. Finally, we discuss the existence of possible common mechanisms that could coordinate their activities.

Rapidly Growing Protein-Centric Technologies to Extensively Identify Protein-RNA Interactions: Application to the Analysis of Co-Transcriptional RNA Processing.

During mRNA transcription, diverse RNA-binding proteins (RBPs) are recruited to RNA polymerase II (RNAP II) transcription machinery. These RBPs bind to distinct sites of nascent RNA to co-transcriptionally operate mRNA processing. Recent studies have revealed a close relationship between transcription and co-transcriptional RNA processing, where one affects the other’s activity, indicating an essential role of protein–RNA interactions for the fine-tuning of mRNA production. Owing to their limited amount in cells, the detection of protein–RNA interactions specifically assembled on the transcribing RNAP II machinery still remains challenging. Currently, cross-linking and immunoprecipitation (CLIP) has become a standard method to detect in vivo protein–RNA interactions, although it requires a large amount of input materials. Several improved methods, such as infrared-CLIP (irCLIP), enhanced CLIP (eCLIP), and target RNA immunoprecipitation (tRIP), have shown remarkable enhancements in the detection efficiency. Furthermore, the utilization of an RNA editing mechanism or proximity labeling strategy has achieved the detection of faint protein–RNA interactions in cells without depending on crosslinking. This review aims to explore various methods being developed to detect endogenous protein–RNA interaction sites and discusses how they may be applied to the analysis of co-transcriptional RNA processing.

Biology of the mRNA Splicing Machinery and Its Dysregulation in Cancer Providing Therapeutic Opportunities.

Dysregulation of messenger RNA (mRNA) processing—in particular mRNA splicing—is a hallmark of cancer. Compared to normal cells, cancer cells frequently present aberrant mRNA splicing, which promotes cancer progression and treatment resistance. This hallmark provides opportunities for developing new targeted cancer treatments. Splicing of precursor mRNA into mature mRNA is executed by a dynamic complex of proteins and small RNAs called the spliceosome. Spliceosomes are part of the supraspliceosome, a macromolecular structure where all co-transcriptional mRNA processing activities in the cell nucleus are coordinated. Here we review the biology of the mRNA splicing machinery in the context of other mRNA processing activities in the supraspliceosome and present current knowledge of its dysregulation in lung cancer. In addition, we review investigations to discover therapeutic targets in the spliceosome and give an overview of inhibitors and modulators of the mRNA splicing process identified so far. Together, this provides insight into the value of targeting the spliceosome as a possible new treatment for lung cancer.