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.

https://www.mdpi.com/1422-0067/22/10/5110/pdf

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

In the cell, RNA exists and functions in a complex with RNA binding proteins (RBPs) that regulate each step of the RNA life cycle from transcription to degradation. Central to this regulation is the role of several molecular chaperones that ensure the correct interactions between RNA and proteins, while aiding the biogenesis of large RNA-protein complexes (ribonucleoproteins or RNPs). Accurate formation of RNPs is fundamentally important to cellular development and function, and its impairment often leads to disease. The survival motor neuron (SMN) protein exemplifies this biological paradigm. SMN is part of a multi-protein complex essential for the biogenesis of various RNPs that function in RNA metabolism. Mutations leading to SMN deficiency cause the neurodegenerative disease spinal muscular atrophy (SMA). A fundamental question in SMA biology is how selective motor system dysfunction results from reduced levels of the ubiquitously expressed SMN protein. Recent clarification of the central role of the SMN complex in RNA metabolism and a thorough characterization of animal models of SMA have significantly advanced our knowledge of the molecular basis of the disease. Here we review the expanding role of SMN in the regulation of gene expression through its multiple functions in RNP biogenesis. We discuss developments in our understanding of SMN activity as a molecular chaperone of RNPs and how disruption of SMN-dependent RNA pathways can contribute to the SMA phenotype.

/pdf/the-smn-complex-at-the-crossroad-between-rna-metabolism-and-chruvnuh.pdf

The SMN Complex at the Crossroad between RNA Metabolism and Neurodegeneration

The spliceosomal snRNP cores, each comprised of a snRNA and a seven-membered Sm ring (D1/D2/F/E/G/D3/B), are assembled by twelve chaperoning proteins in human. However, only six assembly-assisting proteins, ICln and the SMN complex (SMN/Gemin2/Gemin6-8), have been found in Schizosaccharomyces pombe (Sp). Here, we used recombinant proteins to reconstitute the chaperone machinery and investigated the roles of these proteins systematically. We found that, like the human system, the assembly in S. pombe requires ICln and the SMN complex sequentially. However, there are several significant differences. For instance, h_F/E/G forms heterohexamers and heterotrimers, while Sp_F/E/G only forms heterohexamers; h_Gemin2 alone can bind D1/D2/F/E/G, but Sp_Gemin2 cannot. Moreover, we found that Sp_Gemin2 is essential using genetic approaches. These mechanistic studies reveal that these six proteins are necessary and sufficient for Sm core assembly at the molecular level, and enrich our understanding of the chaperone systems in species variation and evolution. 

Mechanism of assembly of snRNP cores assisted by ICln and the SMN complex in fission yeast

U7 snRNP is a multi-subunit endonuclease required for 3’ end processing of metazoan replication-dependent histone pre-mRNAs. In contrast to the spliceosomal snRNPs, U7 snRNP lacks the Sm subunits D1 and D2 and instead contains two related proteins, Lsm10 and Lsm11. The remaining five subunits of the U7 heptameric Sm ring, SmE, F, G, B and D3, are shared with the spliceosomal snRNPs. The pathway that assembles the unique ring of U7 snRNP is unknown. Here, we show that a heterodimer of Lsm10 and Lsm11 tightly interacts with the methylosome, a complex of the arginine methyltransferase PRMT5, MEP50 and pICln known to methylate arginines in the C-terminal regions of the Sm proteins B, D1 and D3 during the spliceosomal Sm ring assembly. Both biochemical and Cryo-EM structural studies demonstrate that the interaction is mediated by PRMT5, which binds and methylates two arginine residues in the N-terminal region of Lsm11. Surprisingly, PRMT5 also methylates an N-terminal arginine in SmE, a subunit that does not undergo this type of modification during the biogenesis of the spliceosomal snRNPs. An intriguing possibility is that the unique methylation pattern of Lsm11 and SmE plays a vital role in the assembly of the U7 snRNP.

/pdf/in-vitro-methylation-of-the-u7-snrnp-subunits-lsm11-and-sme-26yydvhg.pdf

In vitro methylation of the U7 snRNP subunits Lsm11 and SmE by the PRMT5/MEP50/pICln methylosome

Amyotrophic lateral sclerosis (ALS) is linked to the reduction of certain nucleoporins in neurons. Increased nuclear localization of charged multivesicular body protein 7 (CHMP7), a protein involved in nuclear pore surveillance, has been identified as a key factor damaging nuclear pores and disrupting transport. Using CRISPR-based microRaft, followed by gRNA identification (CRaft-ID), we discovered 55 RNA-binding proteins (RBPs) that influence CHMP7 localization, including SmD1, a survival of motor neuron (SMN) complex component. Immunoprecipitation-mass spectrometry (IP-MS) and enhanced crosslinking and immunoprecipitation (CLIP) analyses revealed CHMP7's interactions with SmD1, small nuclear RNAs, and splicing factor mRNAs in motor neurons (MNs). ALS induced pluripotent stem cell (iPSC)-MNs show reduced SmD1 expression, and inhibiting SmD1/SMN complex increased CHMP7 nuclear localization. Crucially, overexpressing SmD1 in ALS iPSC-MNs restored CHMP7's cytoplasmic localization and corrected STMN2 splicing. Our findings suggest that early ALS pathogenesis is driven by SMN complex dysregulation. 

Inhibition of RNA splicing triggers CHMP7 nuclear entry, impacting TDP-43 function and leading to the onset of ALS cellular phenotypes

The assembly of snRNP cores, in which seven Sm proteins, D1/D2/F/E/G/D3/B, form a ring around the nonameric Sm site of snRNAs, is the early step of spliceosome formation and essential to eukaryotes. It is mediated by the PMRT5 and SMN complexes sequentially in vivo. SMN deficiency causes neurodegenerative disease spinal muscular atrophy (SMA). How the SMN complex assembles snRNP cores is largely unknown, especially how the SMN complex achieves high RNA assembly specificity and how it is released. Here we show, using crystallographic and biochemical approaches, that Gemin2 of the SMN complex enhances RNA specificity of SmD1/D2/F/E/G via a negative cooperativity between Gemin2 and RNA in binding SmD1/D2/F/E/G. Gemin2, independent of its N-tail, constrains the horseshoe-shaped SmD1/D2/F/E/G from outside in a physiologically relevant, narrow state, enabling high RNA specificity. Moreover, the assembly of RNAs inside widens SmD1/D2/F/E/G, causes the release of Gemin2/SMN allosterically and allows SmD3/B to join. The assembly of SmD3/B further facilitates the release of Gemin2/SMN. This is the first to show negative cooperativity in snRNP assembly, which provides insights into RNA selection and the SMN complex's release. These findings reveal a basic mechanism of snRNP core assembly and facilitate pathogenesis studies of SMA.

Negative cooperativity between Gemin2 and RNA provides insights into RNA selection and the SMN complex's release in snRNP assembly.

Visualization test strip reading of urine miRNA via cascade selective recognition reactions for non-invasive diagnosis of bladder cancer

The assembly of snRNP cores, in which seven Sm proteins, D1/D2/F/E/G/D3/B, form a ring around the nonameric Sm site of snRNAs, is the early step of spliceosome formation and essential to eukaryotes. It is mediated by the PMRT5 and SMN complexes sequentially in vivo. SMN deficiency causes neurodegenerative disease spinal muscular atrophy (SMA). How the SMN complex assembles snRNP cores is largely unknown, especially how the SMN complex achieves high RNA assembly specificity and how it is released. Here we show, using crystallographic and biochemical approaches, that Gemin2 of the SMN complex enhances RNA specificity of SmD1/D2/F/E/G via a negative cooperativity between Gemin2 and RNA in binding SmD1/D2/F/E/G. Gemin2, independent of its N-tail, constrains the horseshoe-shaped SmD1/D2/F/E/G from outside in a physiologically relevant, narrow state, enabling high RNA specificity. Moreover, the assembly of RNAs inside widens SmD1/D2/F/E/G, causes the release of Gemin2/SMN allosterically and allows SmD3/B to join. The assembly of SmD3/B further facilitates the release of Gemin2/SMN. This is the first to show negative cooperativity in snRNP assembly, which provides insights into RNA selection and the SMN complex9s release. These findings reveal a basic mechanism of snRNP core assembly and facilitate pathogenesis studies of SMA.

https://www.biorxiv.org/content/biorxiv/early/2019/11/14/312124.full.pdf

Negative Cooperativity between Gemin2 and RNA provides Insights into RNA Selection and the SMN Complex’s Release in snRNP Assembly

The assembly of snRNP cores, in which seven Sm proteins, D1/D2/F/E/G/D3/B, form a ring around snRNAs, is the early step of spliceosome formation and essential to eukaryotes. It is mediated by the PMRT5 and SMN complexes sequentially in vivo. The deficiency of SMN causes neurodegenerative disease spinal muscular atrophy (SMA). How the SMN complex assembles snRNP cores in the second phase is largely unknown, especially how the SMN complex achieves stringent RNA specificity, ensuring seven Sm proteins assemble only around snRNAs, by requiring an extra 39-adjacent stem-loop (SL) in addition to a nonameric Sm site RNA (PuAUUUNUGPu) on which snRNP cores can spontaneously form without chaperons in vitro. Moreover, how the SMN complex is released from snRNP cores is unknown. Here we show that Gemin2 of the SMN complex and RNA allosterically and mutually inhibit each other9s binding to SmD1/D2/F/E/G, coupling RNA selection with the SMN complex9s release. Using crystallographic and biochemical approaches, we found that Gemin2 constrains the horseshoe-shaped SmD1/D2/F/E/G in a physiologically relevant, narrow state, which prefers the snRNP-code (both the Sm site and 39-SL)-containing RNA for assembly. Moreover, the assembly of RNA widens SmD1/D2/F/E/G, causes Gemin29s release allosterically and allows SmD3/B to join. By structural analysis we further propose a structural mechanism for the allosteric conformational changes. These findings provide deeper insights into the SMN complex9s mode of action and snRNP assembly, and facilitate potential therapeutic studies of SMA.

https://www.biorxiv.org/content/biorxiv/early/2018/05/02/312124.full.pdf

Negative Cooperativity between Gemin2 and RNA Determines RNA Selection and Release of the SMN Complex in snRNP Assembly

Interconversion and mechanisms between Lsm-type and Sm-type heteroheptameric rings: implications for spliceosome evolution and RNA metabolism

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