U1 snRNP Research Articles

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

U7 snRNP is a multisubunit 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 carboxy-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 amino-terminal region of Lsm11. Surprisingly, PRMT5 also methylates an amino-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.

The fission yeast methyl phosphate capping enzyme Bmc1 guides 2'-O-methylation of the U6 snRNA.

Splicing requires the tight coordination of dynamic spliceosomal RNAs and proteins. U6 is the only spliceosomal RNA transcribed by RNA Polymerase III and undergoes an extensive maturation process. In humans and fission yeast, this includes addition of a 5' γ-monomethyl phosphate cap by members of the Bin3/MePCE family as well as snoRNA guided 2'-O-methylation. Previously, we have shown that the Bin3/MePCE homolog Bmc1 is recruited to the S. pombe telomerase holoenzyme by the LARP7 family protein Pof8, where it acts in a catalytic-independent manner to protect the telomerase RNA and facilitate holoenzyme assembly. Here, we show that Bmc1 and Pof8 are required for the formation of a distinct U6 snRNP that promotes 2'-O-methylation of U6, and identify a non-canonical snoRNA that guides this methylation. We also show that the 5' γ-monomethyl phosphate capping activity of Bmc1 is not required for its role in promoting snoRNA guided 2'-O-methylation, and that this role relies on different regions of Pof8 from those required for Pof8 function in telomerase. Our results are consistent with a novel role for Bmc1/MePCE family members in stimulating 2'-O-methylation and a more general role for Bmc1 and Pof8 in guiding noncoding RNP assembly beyond the telomerase RNP.

Nsp14 of SARS-CoV-2 inhibits mRNA processing and nuclear export by targeting the nuclear cap-binding complex.

To facilitate selfish replication, viruses halt host gene expression in various ways. The nuclear export of mRNA is one such process targeted by many viruses. SARS-CoV-2, the etiological agent of severe acute respiratory syndrome, also prevents mRNA nuclear export. In this study, Nsp14, a bifunctional viral replicase subunit, was identified as a novel inhibitor of mRNA nuclear export. Nsp14 induces poly(A)+ RNA nuclear accumulation and the dissolution/coalescence of nuclear speckles. Genome-wide gene expression analysis revealed the global dysregulation of splicing and 3'-end processing defects of replication-dependent histone mRNAs by Nsp14. These abnormalities were also observed in SARS-CoV-2-infected cells. A mutation introduced at the guanine-N7-methyltransferase active site of Nsp14 diminished these inhibitory activities. Targeted capillary electrophoresis-mass spectrometry analysis (CE-MS) unveiled the production of N7-methyl-GTP in Nsp14-expressing cells. Association of the nuclear cap-binding complex (NCBC) with the mRNA cap and subsequent recruitment of U1 snRNP and the stem-loop binding protein (SLBP) were impaired by Nsp14. These data suggest that the defects in mRNA processing and export arise from the compromise of NCBC function by N7-methyl-GTP, thus exemplifying a novel viral strategy to block host gene expression.

The U1 antisense morpholino oligonucleotide (AMO) disrupts U1 snRNP structure to promote intronic PCPA modification of pre-mRNAs

Functional depletion of the U1 small nuclear ribonucleoprotein (snRNP) with a 25nt U1 AMO (antisense morpholino oligonucleotide) may lead to intronic premature cleavage and polyadenylation of thousands of genes, a phenomenon known as U1 snRNP telescripting; however, the underlying mechanism remains elusive. In this study, we demonstrated that U1 AMO could disrupt U1 snRNP structure both invitro and invivo, thereby affecting the U1 snRNP-RNAP polymerase II interaction. By performing chromatin immunoprecipitation sequencing for phosphorylation of Ser2 and Ser5 of the C-terminal domain of RPB1, the largest subunit of RNAP polymerase II, we showed that transcription elongation was disturbed upon U1 AMO treatment, with a particular high phosphorylation of Ser2 signal at intronic cryptic polyadenylation sites (PASs). In addition, we showed that core 3'processing factors CPSF/CstF are involved in the processing of intronic cryptic PAS. Their recruitment accumulated toward cryptic PASs upon U1 AMO treatment, as indicated by chromatin immunoprecipitation sequencing and individual-nucleotide resolution CrossLinking and ImmunoPrecipitation sequencing analysis. Conclusively, our data suggest that disruption of U1 snRNP structure mediated by U1 AMO provides a key for understanding the U1 telescripting mechanism.

Mouse nuclear RNAi-defective 2 promotes splicing of weak 5' splice sites.

Removal of introns during pre-mRNA splicing, which is central to gene expression, initiates by base pairing of U1 snRNA with a 5' splice site (5'SS). In mammals, many introns contain weak 5'SSs that are not efficiently recognized by the canonical U1 snRNP, suggesting alternative mechanisms exist. Here, we develop a cross-linking immunoprecipitation coupled to a high-throughput sequencing method, BCLIP-seq, to identify NRDE2 (nuclear RNAi-defective 2), and CCDC174 (coiled-coil domain-containing 174) as novel RNA-binding proteins in mouse ES cells that associate with U1 snRNA and 5'SSs. Both proteins bind directly to U1 snRNA independently of canonical U1 snRNP-specific proteins, and they are required for the selection and effective processing of weak 5'SSs. Our results reveal that mammalian cells use noncanonical splicing factors bound directly to U1 snRNA to effectively select suboptimal 5'SS sequences in hundreds of genes, promoting proper splice site choice, and accurate pre-mRNA splicing.

Response to Tofacitinib in an Infant with Aicardi-Goutières Syndrome due to Biallelic Variants in RNU7–1

Aicardi-Goutières syndrome (AGS) is a severe, early-onset neuroinflammatory disease characterized by leukoencephalopathy resulting in developmental delays and neurologic regression. Patients can have extracranial findings including hematologic, cutaneous, and other organ manifestations. AGS is a recognized type I interferonopathy, with evidence of interferon activation in both the central nervous system and blood, leading to attempts to modulate disease using JAnus Kinase (JAK) inhibition.We present a now 1-year-old female who had suspected AGS despite nondiagnostic clinical genetic testing at birth, done for prematurity, failure to thrive requiring enteral tube feeding, leukodystrophy, hypotonia, seizures, hearing loss, and developmental delay. She also has dysmorphisms, including neonatal teeth, rib abnormalities and microcephaly. She was sequentially diagnosed with hepatitis (biopsy findings of mild macrovesicular steatosis, mild interface hepatitis and mildly increased fibrosis), hypertrophic cardiomyopathy, hyponatremia attributed, in part, to adrenal insufficiency, and chronic lung disease from microaspirations. Evaluation at 11 months revealed unremarkable immunoglobulin levels, vaccine titers, immunophenotyping, and proliferation to mitogens and Candida but not tetanus (despite protective titers). An elevated 6-gene interferon score (19.5) was detected in the blood.Given the devastating natural history of this syndrome, she was started on the JAK inhibitor tofacitinib age 8 months. After starting tofacitinib, she had notably decreased clinical symptoms, improved tone and milestones. Her transaminitis resolved. A repeat interferon score after 4 months of treatment showed improvement (15.9). She has tolerated common viral infections on immune suppression.Under the Undiagnosed Diseases Network, clinical trio genome sequencing was re-analyzed revealing compound heterozygous variants in the non-coding RNA RNU7–1, a newly identified cause of previously genetically unexplained AGS (AGS9). This results in impairment of U7 snRNP function leading to misprocessing of pre-mRNA and driving an interferon response. The mortality of AGS9 is higher than other AGS genotypes, with cardiac and liver involvement being more common.Revelation of RNU7–1 mutations in this case underscores that diagnostic workup for genetically unexplained AGS should include targeted evaluation of RNU7–1. The impact of early initiation of JAK inhibition in patients with AGS on long-term outcomes remains to be determined, but tofacitinib, uniquely available in liquid formulation, is one option.

Structural insights into recognition of SL4, the UUCG stem-loop, of human U1 snRNA by the ubiquitin-like domain, including the C-terminal tail in the SF3A1 subunit of U2 snRNP.

The pre-spliceosomal complex involves interactions between U1 and U2 snRNPs, where a ubiquitin-like domain (ULD) of SF3A1, a component of U2 snRNP, binds to the stem-loop 4 (SL4; the UUCG tetraloop) of U1 snRNA in U1 snRNP. Here, we reported the 1.80 Å crystal structure of human SF3A1 ULD (ULDSF3A1) complexed with SL4. The structural part of ULDSF3A1 (res. 704-785) adopts a typical β-grasp fold with a topology of β1-β2-α1-310a-β3-β4-310b-β5, closely resembling that of ubiquitin, except for the length and structure of the β1/β2 loop. A patch on the surface formed by three ULDSF3A1-specific residues, Lys756 (β3), Phe763 (β4), and Lys765 (following β4), contacts the canonical UUCG tetraloop structure. In contrast, the directly following C-terminal tail composed of 786KERGGRKK793 was essentially stretched. The main or side chains of all the residues interacted with the major groove of the stem helix; the RGG residues adopted a peculiar conformation for RNA recognition. These findings were confirmed by mutational studies using bio-layer interferometry. Collectively, a unique combination of the β-grasp fold and the C-terminal tail constituting ULDSF3A1 is required for the SL4-specific binding. This interaction mode also suggests that putative post-translational modifications, including ubiquitination in ULDSF3A1, directly inhibit SL4 binding.

High BRCA1 expression is an independent prognostic biomarker in LUAD and correlates with immune infiltration

High BRCA1 expression is an independent prognostic biomarker in LUAD and correlates with immune infiltration

U1 snRNP increases RNA Pol II elongation rate to enable synthesis of long genes

The expansion of introns within mammalian genomes poses a challenge for the production of full-length messenger RNAs (mRNAs), with increasing evidence that these long AT-rich sequences present obstacles to transcription. Here, we investigate RNA polymerase II (RNAPII) elongation at high resolution in mammalian cells and demonstrate that RNAPII transcribes faster across introns. Moreover, we find that this acceleration requires the association of U1 snRNP (U1) with the elongation complex at 5' splice sites. The role of U1 to stimulate elongation rate through introns reduces the frequency of both premature termination and transcriptional arrest, thereby dramatically increasing RNA production. We further show that changes in RNAPII elongation rate due to AT content and U1 binding explain previous reports of pausing or termination at splice junctions and the edge of CpG islands. We propose that U1-mediated acceleration of elongation has evolved to mitigate the risks that long AT-rich introns pose to transcript completion.

Phylogenetic Analysis of Spliceosome SF3a2 in Different Plant Species

The formation of mature mRNA requires cutting introns and splicing exons. The occurrence of splicing involves the participation of the spliceosome. Common spliceosomes mainly include five snRNPs: U1, U2, U4/U6, and U5. SF3a2, an essential component of spliceosome U2 snRNP, participates in splicing a series of genes. There is no definition of SF3a2 in plants. The paper elaborated on SF3a2s from a series of plants through protein sequence similarity. We constructed the evolutionary relationship of SF3a2s in plants. Moreover, we analyzed the similarities and differences in gene structure, protein structure, the cis-element of the promoter, and expression pattern; we predicted their interacting proteins and constructed their collinearity. We have preliminarily analyzed SF3a2s in plants and clarified the evolutionary relationship between different species; these studies can better serve for in-depth research on the members of the spliceosome in plants.

Mechanisms of the RNA helicases DDX42 and DDX46 in human U2 snRNP assembly

Three RNA helicases – DDX42, DDX46 and DHX15 – are found to be associated with human U2 snRNP, but their roles and mechanisms in U2 snRNP and spliceosome assembly are insufficiently understood. Here we report the cryo-electron microscopy (cryo-EM) structures of the DDX42-SF3b complex and a putative assembly precursor of 17S U2 snRNP that contains DDX42 (DDX42-U2 complex). DDX42 is anchored on SF3B1 through N-terminal sequences, with its N-plug occupying the RNA path of SF3B1. The binding mode of DDX42 to SF3B1 is in striking analogy to that of DDX46. In the DDX42-U2 complex, the N-terminus of DDX42 remains anchored on SF3B1, but the helicase domain has been displaced by U2 snRNA and TAT-SF1. Through in vitro assays, we show DDX42 and DDX46 are mutually exclusive in terms of binding to SF3b. Cancer-driving mutations of SF3B1 target the residues in the RNA path that directly interact with DDX42 and DDX46. These findings reveal the distinct roles of DDX42 and DDX46 in assembly of 17S U2 snRNP and provide insights into the mechanisms of SF3B1 cancer mutations.

Building a quantitative and predictive model of 5'SS selection by human U1 snRNP using RNA-map.

Alternative splicing of pre-mRNA greatly expands the protein coding potential of our genome and accounts for extensive cell and tissue-specific variation in gene expression. Spliceosome assembly depends on the recognition of the 5′ splice site (5′SS) at the exon-intron boundary by the U1 small nuclear ribonucleoprotein (U1 snRNP). Critically, mutations in either U1 or the 5′SS can lead to human disease, including cancer. Despite decades of investigation, accurately predicting 5′SS selection and alternative splicing outcomes is difficult, suggesting new approaches are needed. Here, we present our progress in developing a quantitative and predictive model of 5′SS selection by human U1 snRNP grounded in biochemical understanding. We used the RNA on a massively parallel array (RNA-MaP) platform to measure equilibrium binding constants of reconstituted human U1 snRNP to >60,000 unique RNAs. Our designed RNA library features all possible GU and GC variants in addition to thousands of identified mutations, allowing us to build a comprehensive and predictive model of 5′SS/U1 interactions at the transcriptome scale. Overall, our results will provide a first principles understanding of 5′SS selection and will serve as a foundation for future work to systematically determine the effects of other splicing factors and splice site usage in vivo.

A kinetic mechanism for U6 snRNP biogenesis obtained by single molecule fluorescence colocalization microscopy.

Pre-mRNA splicing is the process of removing introns and forming a mature mRNA product by the spliceosome. The U6 small nuclear RNA ribonucleoprotein (snRNP) is an essential component of the splicing machinery and is composed of the U6 snRNA, Prp24, and Lsm2-8 heteroheptameric ring. In the complex, there are both RNA-protein and protein-protein interactions that play critical roles in snRNP assembly. Here, we are studying how the Lsm2-8 ring is recruited to the U6 snRNA in real-time using single-molecule fluorescence colocalization microscopy (CoSMoS). By using fluorescently labeled RNAs and proteins, we have analyzed single molecule reaction trajectories to determine the initial kinetic parameters for snRNP formation and how these change due to post-transcriptional modification of the RNA. We have also elucidated the function of highly-conserved protein domains in Prp24 in both splicing factor recruitment and stabilization of the RNP. Overall, our single-molecule results allow us to propose a kinetic mechanism for snRNP assembly and elucidate how assembly can proceed efficiently in both the presence and absence of RNA modifications.

SART3 associates with a post-splicing complex.

SART3 is a multifunctional protein that acts in several steps of gene expression, including assembly and recycling of the spliceosomal U4/U6 small nuclear ribonucleoprotein particle (snRNP). In this work, we provide evidence that SART3 associates via its N-terminal HAT domain with the 12S U2 snRNP. Further analysis showed that SART3 associates with the post-splicing complex containing U2 and U5 snRNP components. In addition, we observed an interaction between SART3 and the RNA helicase DHX15, which disassembles post-splicing complexes. Based on our data, we propose a model that SART3 associates via its N-terminal HAT domain with the post-splicing complex, where it interacts with U6 snRNA to protect it and to initiate U6 snRNA recycling before a next round of splicing.

Reovirus μ2 Protein Impairs Translation to Reduce U5 snRNP Protein Levels

Mammalian orthoreovirus (MRV) is a double-stranded RNA virus from the Reoviridae family that infects a large range of mammals, including humans. Recently, studies have shown that MRV alters cellular alternative splicing (AS) during viral infection. The structural protein μ2 appears to be the main determinant of these AS modifications by decreasing the levels of U5 core components EFTUD2, PRPF8, and SNRNP200 during infection. In the present study, we investigated the mechanism by which μ2 exerts this effect on the U5 components. Our results revealed that μ2 has no impact on steady-state mRNA levels, RNA export, and protein stability of these U5 snRNP proteins. However, polysome profiling and metabolic labeling of newly synthesized proteins revealed that μ2 exerts an inhibitory effect on global translation. Moreover, we showed that μ2 mutants unable to accumulate in the nucleus retain most of the ability to reduce PRPF8 protein levels, indicating that the effect of μ2 on U5 snRNP components mainly occurs in the cytoplasm. Finally, co-expression experiments demonstrated that μ2 suppresses the expression of U5 snRNP proteins in a dose-dependent manner, and that the expression of specific U5 snRNP core components have different sensitivities to μ2's presence. Altogether, these results suggest a novel mechanism by which the μ2 protein reduces the levels of U5 core components through translation inhibition, allowing this viral protein to alter cellular AS during infection.

A genetic screen in Arabidopsis reveals the identical roles for RBP45d and PRP39a in 5' cryptic splice site selection.

Cryptic splice sites in eukaryotic genome are generally dormant unless activated by mutation of authentic splice sites or related splicing factors. How cryptic splice sites are used remains unclear in plants. Here, we identified two cryptic splicing regulators, RBP45d and PRP39a that are homologs of yeast U1 auxiliary protein Nam8 and Prp39, respectively, via genetic screening for suppressors of the virescent sot5 mutant, which results from a point mutation at the 5' splice site (5' ss) of SOT5 intron 7. Loss-of-function mutations in RBP45d and PRP39a significantly increase the level of a cryptically spliced variant that encodes a mutated but functional sot5 protein, rescuing sot5 to the WT phenotype. We furtherly demonstrated that RBP45d and PRP39a interact with each other and also with the U1C, a core subunit of U1 snRNP. We found that RBP45d directly binds to the uridine (U)-rich RNA sequence downstream the 5' ss of SOT5 intron 7. However, other RBP45/47 members do not function redundantly with RBP45d, at least in regulation of cryptic splicing. Taken together, RBP45d promotes U1 snRNP to recognize the specific 5' ss via binding to intronic U-rich elements in plants.

U5 snRNP Core Proteins Are Key Components of the Defense Response against Viral Infection through Their Roles in Programmed Cell Death and Interferon Induction.

The spliceosome is a massive ribonucleoprotein structure composed of five small nuclear ribonucleoprotein (snRNP) complexes that catalyze the removal of introns from pre-mature RNA during constitutive and alternative splicing. EFTUD2, PRPF8, and SNRNP200 are core components of the U5 snRNP, which is crucial for spliceosome function as it coordinates and performs the last steps of the splicing reaction. Several studies have demonstrated U5 snRNP proteins as targeted during viral infection, with a limited understanding of their involvement in virus-host interactions. In the present study, we deciphered the respective impact of EFTUD2, PRPF8, and SNRNP200 on viral replication using mammalian reovirus as a model. Using a combination of RNA silencing, real-time cell analysis, cell death and viral replication assays, we discovered distinct and partially overlapping novel roles for EFTUD2, PRPF8, and SNRNP200 in cell survival, apoptosis, necroptosis, and the induction of the interferon response pathway. For instance, we demonstrated that EFTUD2 and SNRNP200 are required for both apoptosis and necroptosis, whereas EFTUD2 and PRPF8 are required for optimal interferon response against viral infection. Moreover, we demonstrated that EFTUD2 restricts viral replication, both in a single cycle and multiple cycles of viral replication. Altogether, these results establish U5 snRNP core components as key elements of the cellular antiviral response.

Elongator and the role of its subcomplexes in human diseases.

The Elongator complex was initially identified in yeast, and a variety of distinct cellular functions have been assigned to the complex. In the last decade, several research groups focussed on dissecting its structure, tRNA modification activity and role in translation regulation. Recently, Elongator emerged as a crucial factor for various human diseases, and its involvement has triggered a strong interest in the complex from numerous clinical groups. The Elongator complex is highly conserved among eukaryotes, with all six subunits (Elp1-6) contributing to its stability and function. Yet, recent studies have shown that the two subcomplexes, namely the catalytic Elp123 and accessory Elp456, may have distinct roles in the development of different neuronal subtypes. This Commentary aims to provide a brief overview and new perspectives for more systematic efforts to explore the functions of the Elongator in health and disease.

Proteogenomic Analysis of SF3B1mut CLL Reveals Altered Protein Signaling Cascades and Metabolomic Pathways

Although mutation in the U2 snRNP spliceosome component SF3B1 has been appreciated as a common driver of chronic lymphocytic leukemia (CLL) for over a decade, we still know little of its mechanism of action. Prior transcriptome-based characterizations have demonstrated widespread changes in alternative splicing in SF3B1mut CLL. We hypothesized that protein level characterization could potentially yield fresh insights on alternative expression of proteins and on the impact of altered signaling pathways in patients bearing this mutation. To this end, we assembled CLL specimens from a cohort of 105 patients enrolled on the German CLL Study Group (GCLLSG) trials CLL8 and CLL11, collected prior to therapy. Of these, 52 patients expressed SF3B1wt and 53 patients harbored clonal SF3B1mut (CCF>0.8); 25 of these were the K700E mutation. Additionally, we characterized these samples by WES (available for 97 patients) and RNA-Seq (available for 96 patients). Proteomes and phosphoproteomes from all 105 samples were analyzed using a multiplexed isobaric-tagged (TMT) high resolution mass spectrometry (MS) workflow. Briefly, cells were lysed and proteins were digested into peptides with LysC and trypsin. Protein yield per sample ranged from 20 μg to 300 μg. Peptides were labeled with TMT-11 reagent and combined into 11 multiplexes while accounting for input material availability and randomizing for SF3B1 mutation, IGHV status and ATM/TP53 mutation. To enable cross TMT-plex comparison, we constructed a common reference consisting of 40 samples with highest protein amount and an equal representation between SF3B1wt and SF3B1mut samples. Each plex was fractionated into 24 fractions by high pH reversed phase separation and 500 ng protein per fraction were analyzed by MS (Orbitrap Exploris, Thermo Fisher Scientific). For phosphoproteome analysis, peptides were pooled into 12 fractions and enriched by IMAC prior to MS analysis. Spectra were analyzed by Spectrum Mill v7.1 (Broad Institute) and searched against a human reference proteome database supplemented with non-canonical ORFs supported by Riboseq evidence of translation (nuORFdb v1.0). Patient specific proteins containing single amino variants, indels, and splice isoforms, derived from WES and RNA-Seq, were included in the search database. We obtained a total of 10,419 proteins, with a median of 8,882 proteins per TMT-plex and 6,969 proteins that were quantifiable across all samples. The phosphoproteome analysis identified 32,410 sites; while each TMT-plex yielded a median of 19,380 phosphosites per plex, 5,810 were quantifiable across all patients. We integrated proteome data with patient specific mutation calls, RNA-Seq and copy number alteration (CNA) data using Panoply, a cloud-based platform for proteogenomic data analysis (Broad Institute). We confirmed the impact of chr11 and chr13 deletion as well as chr12 amplification on protein expression. We readily identified peptides spanning the SF3B1mut amino acid regions in corresponding samples. By unsupervised clustering, samples were grouped by SF3B1 status on the CNA and proteome level, a trend not observed based on RNA or phosphoproteome analysis. Gene set enrichment analysis revealed enrichment of TNFA and PI3K signaling in the SF3B1wt clusters, while the SF3B1mut associated clusters showed a strong association with metabolism changes. This analysis was highly concordant with a parallel interrogation comparing proteins expressed in SF3B1wt CLL versus SF3B1mut CLL. Here 1,955 proteins and 1,737 phosphosites were significantly altered (5% FDR). Proteins involved in B cell receptor and MAP kinase signaling were highly expressed in SF3B1wt samples, while again amino acid metabolism pathways as well as oxidative phosphorylation were upregulated in SF3B1mut samples. To more deeply investigate the role of metabolism pathways in SF3B1mut CLL, we subjected 6 independent CLL samples to MS-based metabolomics analysis. Inosine (P=0.029) and guanosine (P=0.047) were lower in the SF3B1mut extracts (P=0.001), while acylcarnitines (P=0.0004) were increased, indicating fatty acid oxidation as a preferred energy source in SF3B1mut CLL. Altogether, our study reveals fresh evidence of altered energy metabolism in SF3B1mut CLL. Ongoing studies focus on defining the mechanism by which SF3B1mut exerts these metabolic changes and novel therapeutic opportunities afforded by these findings.

Phylogenetic analysis and stress response of the plant U2 small nuclear ribonucleoprotein B″ gene family

BackgroundAlternative splicing (AS) is an important channel for gene expression regulation and protein diversification, in addition to a major reason for the considerable differences in the number of genes and proteins in eukaryotes. In plants, U2 small nuclear ribonucleoprotein B″ (U2B″), a component of splicing complex U2 snRNP, plays an important role in AS. Currently, few studies have investigated plant U2B″, and its mechanism remains unclear.ResultPhylogenetic analysis, including gene and protein structures, revealed that U2B″ is highly conserved in plants and typically contains two RNA recognition motifs. Subcellular localisation showed that OsU2B″ is located in the nucleus and cytoplasm, indicating that it has broad functions throughout the cell. Elemental analysis of the promoter region showed that it responded to numerous external stimuli, including hormones, stress, and light. Subsequent qPCR experiments examining response to stress (cold, salt, drought, and heavy metal cadmium) corroborated the findings. The prediction results of protein–protein interactions showed that its function is largely through a single pathway, mainly through interaction with snRNP proteins.ConclusionU2B″ is highly conserved in the plant kingdom, functions in the nucleus and cytoplasm, and participates in a wide range of processes in plant growth and development.