FIG 1 - available via license: Creative Commons Attribution 4.0 International
Content may be subject to copyright.
Analysis of a structurally altered form of SAF. Nucleic acid sequence of a novel form of SAF cDNA, designated as SAF-2, from HeLa cells is shown. The initiator ATG codon is depicted as white box. Comparative analysis of SAF-2 sequence with that of SAF-1, a homolog of rabbit SAF-1, is shown. Dots indicate common sequence between SAF-1 and SAF-2. The unique coding triplets of SAF-1 and SAF-2 are shown by underlines and overlines, respectively. Polyalanine, polyproline, and zinc finger domains are indicated. Termination codons of SAF-1 and SAF-2 are indicated by shaded and black boxes.
Source publication
Serum amyloid A-activating factor-1 (SAF-1), a Cys(2)His(2)-type zinc finger transcription factor, regulates inflammation-induced expression of serum amyloid A protein that is linked to the pathogenesis of reactive amyloidosis, rheumatoid arthritis, and atherosclerosis. Here we report the identification of a novel splice variant, SAF-2, of the SAF...
Citations
... About 6.2% of non-syndromic males with genitourinary anomalies have copy number variants that encompass MAZ [60]. MAZ can act as a transcriptional initiator or terminator; among MAZ targets are the promotor regions of WT1, MYC and Sp1 [61,62]. MAZ regulation of Wnt morphogens is implicated as required for normal genitourinary development [60]. ...
Genitourinary development is a delicately orchestrated process that begins in the embryo. Once complete, the genitourinary system is a collection of functionally disparate organs spread throughout the abdominal and pelvic regions. These distinct organs are interconnected through an elaborate duct system which aggregates the organs' products to a common exit point. The complicated nature of the genitourinary system makes it highly susceptible to developmental disruptions that produce anomalies. In fact, genitourinary anomalies are among the most common class of human birth defects. Aside from congenital anomalies of the kidney and urinary tract (CAKUT), for males, these birth defects can also occur in the penis (hypospadias) and testis (cryptorchism), which impact male fertility and male mental health. As genetic technology has advanced, it has become clear that a subset of cases of genitourinary birth defects are due to gene variation causing dosage changes in critical regulatory genes. Here we first review the parallels between human and mouse genitourinary development. We then demonstrate how translational research leverages mouse models of human gene variation cases to advance mechanistic understanding of causation in genitourinary birth defects. We close with a view to the future highlighting upcoming technologies that will provide a deeper understanding of gene variation affecting regulation of genitourinary development, which should ultimately advance treatment options for patients.
... Similarly, KRAS G12V cells differentially splice 409 events compared to KRAS WT cells including 232 cassette exons (Fig. 4 A). Of 7568 differentially spliced events observed in total, 5233 were observed only once in the experiment, splicing of MAZ therefore leads to two distinct protein isoforms with different C termini, which have been shown to have different DNA-binding properties [49]. ...
... Among the most recurrently differentially spliced transcripts in these multiple lung cell contexts was alternative splicing of MAZ, which we validated by quantitative RT-PCR. MAZ encodes a transcription factor which has been shown to regulate expression of KRAS and HRAS [49]. MAZ is a member of the MAZ-like family of transcription factors which also includes VEZF1 [51]. ...
Background:
Alternative RNA splicing is widely dysregulated in cancers including lung adenocarcinoma, where aberrant splicing events are frequently caused by somatic splice site mutations or somatic mutations of splicing factor genes. However, the majority of mis-splicing in cancers is unexplained by these known mechanisms. We hypothesize that the aberrant Ras signaling characteristic of lung cancers plays a role in promoting the alternative splicing observed in tumors.
Methods:
We recently performed transcriptome and proteome profiling of human lung epithelial cells ectopically expressing oncogenic KRAS and another cancer-associated Ras GTPase, RIT1. Unbiased analysis of phosphoproteome data identified altered splicing factor phosphorylation in KRAS-mutant cells, so we performed differential alternative splicing analysis using rMATS to identify significantly altered isoforms in lung epithelial cells. To determine whether these isoforms were uniquely regulated by KRAS, we performed a large-scale splicing screen in which we generated over 300 unique RNA sequencing profiles of isogenic A549 lung adenocarcinoma cells ectopically expressing 75 different wild-type or variant alleles across 28 genes implicated in lung cancer.
Results:
Mass spectrometry data showed widespread downregulation of splicing factor phosphorylation in lung epithelial cells expressing mutant KRAS compared to cells expressing wild-type KRAS. We observed alternative splicing in the same cells, with 2196 and 2416 skipped exon events in KRASG12V and KRASQ61H cells, respectively, 997 of which were shared (p < 0.001 by hypergeometric test). In the high-throughput splicing screen, mutant KRAS induced the greatest number of differential alternative splicing events, second only to the RNA binding protein RBM45 and its variant RBM45M126I. We identified ten high confidence cassette exon events across multiple KRAS variants and cell lines. These included differential splicing of the Myc Associated Zinc Finger (MAZ). As MAZ regulates expression of KRAS, this splice variant may be a mechanism for the cell to modulate wild-type KRAS levels in the presence of oncogenic KRAS.
Conclusion:
Proteomic and transcriptomic profiling of lung epithelial cells uncovered splicing factor phosphorylation and mRNA splicing events regulated by oncogenic KRAS. These data suggest that in addition to widespread transcriptional changes, the Ras signaling pathway can promote post-transcriptional splicing changes that may contribute to oncogenic processes.
... The copyright holder for this preprint (which this version posted May 20, 2022. alternative C terminus (Ray et al. 2002). In both AALE and A549 cells, expression of KRAS mut 1 resulted in an increase in exon V' inclusion (∆PSI = .14 ...
Background
Alternative RNA splicing is widely dysregulated in cancers including lung adenocarcinoma, where aberrant splicing events are frequently caused by somatic splice site mutations or somatic mutations of splicing factor genes. However, the majority of mis-splicing in cancers is unexplained by these known mechanisms. We hypothesize that the aberrant Ras signaling characteristic of lung cancers plays a role in promoting the alternative splicing observed in tumors.
Methods
We recently performed transcriptome and proteome profiling of human lung epithelial cells ectopically expressing oncogenic KRAS and another cancer-associated Ras GTPase, RIT1. Unbiased analysis of phosphoproteome data identified altered splicing factor phosphorylation in KRAS-mutant cells, so we performed differential alternative splicing analysis using rMATS to identify significantly altered isoforms in lung epithelial. To determine whether these isoforms were uniquely regulated by KRAS, we performed a large-scale splicing screen in which we generated over 300 unique RNA sequencing profiles of isogenic A549 lung adenocarcinoma cells ectopically expressing 75 different wild-type or variant alleles across 28 genes implicated in lung cancer.
Results
Mass spectrometry data showed widespread downregulation of splicing factor phosphorylation in lung epithelial cells expressing mutant KRAS compared to cells expressing wild-type KRAS. We observed alternative splicing in the same cells, with 2196 and 2416 skipped exon events in KRAS G12V and KRAS Q61H cells, respectively, 997 of which were shared (p < 0.001 by hypergeometric test). In the high-throughput splicing screen, mutant KRAS induced the greatest number of differential alternative splicing events, second only to the RNA binding protein RBM45 and its mutant allele RBM45 M126I . We identified ten high confidence cassette exon events across multiple KRAS variants and cell lines. These included differential splicing of the Myc Associated Zinc Finger (MAZ). As MAZ regulates expression of KRAS, this splice variant may be a mechanism for the cell to modulate wild-type KRAS levels in the presence of oncogenic KRAS.
Conclusion
Proteomic and transcriptomic profiling of lung epithelial cells uncovered splicing factor phosphorylation and mRNA splicing events regulated by oncogenic KRAS. These data suggest that in addition to widespread transcriptional changes, Ras signaling pathways in cancer promote post-transcriptional splicing changes that may contribute to oncogenic processes.
... GADD45A also exhibits a changed splicing pattern in cancer cells, with the shorter splice form inhibiting cell cycle progression during stress Salvador et al. 2013;Carbonell et al. 2019). The longer MAZ exon 5 variant has been shown to bind more strongly to DNA and inhibit the activation of inflammatory genes induced by the shorter isoform (Ray et al. 2002;Triner et al. 2018). These examples indicate that SWI/SNF complexes affect the balances between splice variants with different functions, and provide an additional way for SWI/SNF complexes to dysregulate targets during cancer progression. ...
BRG1 and BRM are ATPase core subunits of the human SWI/SNF chromatin remodelling complexes mainly associated with transcriptional initiation. They also have a role in alternative splicing, which has been shown for BRM-containing SWI/SNF complexes at a few genes. Here, we have identified a subset of genes which harbour alternative exons that are affected by SWI/SNF ATPases by expressing the ATPases BRG1 and BRM in C33A cells, a BRG1- and BRM-deficient cell line, and analysed the effect on splicing by RNA sequencing. BRG1- and BRM-affected sub-sets of genes favouring both exon inclusion and exon skipping, with only a minor overlap between the ATPase. Some of the changes in alternative splicing induced by BRG1 and BRM expression did not require the ATPase activity. The BRG1-ATPase independent included exons displayed an exon signature of a high GC content. By investigating three genes with exons affected by the BRG-ATPase-deficient variant, we show that these exons accumulated phosphorylated RNA pol II CTD, both serine 2 and serine 5 phosphorylation, without an enrichment of the RNA polymerase II. The ATPases were recruited to the alternative exons, together with both core and signature subunits of SWI/SNF complexes, and promoted the binding of RNA binding factors to chromatin and RNA at the alternative exons. The interaction with the nascent RNP, however, did not reflect the association to chromatin. The hnRNPL, hnRNPU and SAM68 proteins associated with chromatin in cells expressing BRG1 and BRM wild type, but the binding of hnRNPU to the nascent RNP was excluded. This suggests that SWI/SNF can regulate alternative splicing by interacting with splicing-RNA binding factor and influence their binding to the nascent pre-mRNA particle.
... Combined with the opening chromatin peaks around MIR9-1HG and potential binding motifs of TFs, three TFs, MAZ, TCF4 and ASCL1, were selected from 8 candidates (Fig. 5f). MAZ has been reported as a transcriptional activator binding protein, 89 suggesting that MAZ would support the expression of MIR9-1HG. The binding motif of TCF4, a TF involved in the initiation of neural differentiation, 90,91 was mapped to the open chromatin peaks around the transcription start site of MIR9-1HG (Fig. 5). ...
The differences in size and function between primate and rodent brains, and the association of disturbed excitatory/inhibitory balance with many neurodevelopmental disorders highlight the importance to study primate ganglionic eminences (GEs) development. Here we used single-cell RNA and ATAC sequencing to characterize the emergence of cell diversity in monkey and human GEs where most striatal and cortical interneurons are generated. We identified regional and temporal diversity among progenitor cells which give rise to a variety of interneurons. These cells are specified within the primate GEs by well conserved gene regulatory networks, similar to those identified in mice. However, we detected, in human, several novel regulatory pathways or factors involved in the specification and migration of interneurons. Importantly, comparison of progenitors between our human and published mouse GE datasets led to the discovery and confirmation of outer radial glial cells in GEs in human cortex. Our findings reveal both evolutionarily conservative and nonconservative regulatory networks in primate GEs, which may contribute to their larger brain sizes and more complex neural networks compared with mouse.
... It is noteworthy that stem cells significantly differ from differentiated cells by the expression levels of various transcription factors (such as pluripotency factors OCT4, SOX2, NANOG) [116][117][118][119][120]. In silico analysis revealed multiple loci inside ITR of AAV which can bind different transcription factors (such as Zic1, YY-1, MAZ, NSCs, C/EBPβ, WT1, Sp1, P53, and Pax-6) [121][122][123][124][125][126][127][128][129][130][131][132][133][134][135][136][137]. Moreover, AAV ITRs can act as promoters [138,139] and influence transgene expression. ...
Gene therapy of genetically determined diseases, including some pathologies of the respiratory system, requires an efficient method for transgene delivery. Recombinant adeno-associated viral (rAAV) vectors are well studied and employed in gene therapy, as they are relatively simple and low immunogenic and able to efficiently transduce eukaryotic cells. To date, many natural and artificial (with modified capsids) AAV serotypes have been isolated, demonstrating preferential tropism toward different tissues and cells in accordance with the prevalent receptors on the cell surface. However, rAAV-mediated delivery is not strictly specific due to wide tropism of some viral serotypes. Thus, the development of the methods allowing modulating specificity of these vectors could be beneficial in some cases. This review describes various approaches for retargeting rAAV to respiratory cells, for example, using different types of capsid modifications and regulation of a transgene expression by tissue-specific promoters. Part of the review is devoted to the issues of transduction of stem and progenitor lung cells using AAV, which is a complicated task today.
... GADD45A also exhibits a changed splicing pattern in cancer cells, with the shorter splice form inhibiting cell cycle progression during stress Salvador et al., 2013;Carbonell et al., 2019). The longer MAZ exon 5 variant has been shown to inhibit the activation of inflammatory genes by the shorter MAZ isoform by binding more strongly to DNA (Ray et al., 2002;Triner et al., 2017). These examples indicate that SWI/SNF complexes affect the balances between splice variants with different functions, which may be an additional way that SWI/SNF targets are dysregulated during cancer progression. ...
BRG1 and BRM are ATPase core subunits of the human SWI/SNF chromatin remodelling complexes. The function of the SWI/SNF complexes in transcriptional initiation has been well studied, while a function in alternative splicing has only been studied for a few cases for BRM-containing SWI/SNF complexes. Here, we have expressed BRG1 in C33A cells, a BRG1 and BRM-deficient cell line, and we have analysed the effects on the transcriptome by RNA sequencing. We have shown that BRG1 expression affects the splicing of a subset of genes. For some, BRG1 expression favours exon inclusion and for others, exon skipping. Some of the changes in alternative splicing induced by BRG1 expression do not require the ATPase activity of BRG1. Among the exons regulated through an ATPase-independent mechanism, the included exons had signatures of high GC-content and lacked a positioned nucleosome at the exon. By investigating three genes in which the expression of either wild-type BRG1 or a BRG1-ATPase-deficient variant favoured exon inclusion, we showed that expression of the ATPases promotes the local recruitment of RNA binding factors to chromatin and RNA in a differential manner. The hnRNPL, hnRNPU and SAM68 proteins associated to chromatin in C33A cells expressing BRG1 or BRM, but their association with RNA varied. We propose that SWI/SNF can regulate alternative splicing by interacting with splicing-RNA binding factor and altering their binding to the nascent pre-mRNA, which changes RNP structure.
Author summary
Splicing, in particular alternative splicing, is a combinatorial process which involves splicing factor complexes and many RNA binding splicing regulatory proteins in different constellations. Most splicing events occur during transcription, which also makes the DNA sequence, the chromatin state and the transcription rate at the exons important components that influence the splicing outcome. We show here that the ATP-dependent chromatin remodelling complex SWI/SNF influences the interactions of splicing regulatory factors with RNA during transcription on certain exons that have a high GC-content. The splicing on this type of exon rely on the ATPase BRG1 and favour inclusion of alternative exons in an ATP-independent manner. SWI/SNF complexes are known to alter the chromatin structure at promoters in transcription initiation, and have been previously shown to alter the transcription rate or nucleosome position in splicing. Our results suggests a further mechanism for chromatin remodelling proteins in splicing: to change the interaction patterns of RNA binding splicing regulatory factors at alternative exons to alter the splicing outcome.
... The MAZ-2 transcript is generated by an alternative splicing that results in the insertion of a new exon originating from the noncoding sequences of the intron 4. This transcript gives rise to the MAZ-2 isoform, which is a 493 amino-acids long protein with distinct carboxyl-terminus which contains two additional zinc-finger domains (Ray et al., 2002). The MAZ-2 isoform is reported to have a higher DNA-binding activity and to act as a negative regulator of MAZ-1 function (Ray et al., 2002). ...
... This transcript gives rise to the MAZ-2 isoform, which is a 493 amino-acids long protein with distinct carboxyl-terminus which contains two additional zinc-finger domains (Ray et al., 2002). The MAZ-2 isoform is reported to have a higher DNA-binding activity and to act as a negative regulator of MAZ-1 function (Ray et al., 2002). The MAZ-3 transcript is expressed at very low levels under normal physiological condition in various tissues, but is highly expressed during inflammation. ...
Myc-associated zinc finger protein (MAZ), also known as serum amyloid A-activating factor 1 (SAF1), Pur-1 or Zif87, is ubiquitously expressed in various tissues. MAZ is a transcription factor with six Cys2His2-type zinc finger motifs at the carboxyl-terminus that interact with a permutation of the GGGAGGG sequence motif present in GC-rich promoter regions of target genes, likely through DNA unfolding of G-quadruplex structures to modulate gene expressions. MAZ is also suggested to participate in transcription termination and polyadenylation. Deregulated expression of MAZ is reported to correlate with various tissue malignancies that include the breast, thyroid, hepatocellular and urothelial cancers. © 2018 Atlas of Genetics and Cytogenetics in Oncology and Haematology.
... MAZ has three splice variants (MAZ1, MAZ2, and MAZ3). MAZ splice variants 1 and 3 are activated by inflammation, whereas MAZ2 is anti-inflammatory (8,9). Our previous work has identified an essential role for MAZ in hypoxia-driven inflammatory responses in both colitis and colon cancer (10). ...
Myc-associated zinc finger (MAZ) is a transcription factor highly upregulated in chronic inflammatory disease and several human cancers. In the current study, we found that MAZ protein is highly expressed in human ulcerative colitis and colon cancer. However, the precise role for MAZ in the progression of colitis and colon cancer is not well defined. To determine the function of MAZ, a novel mouse model of intestine epithelial specific MAZ overexpression was generated. Expression of MAZ in intestinal epithelial cells was sufficient to enhance inflammatory injury in two complementary models of colitis. Moreover, MAZ expression increased tumorigenesis in an in vivo model of inflammation-induced colon cancer and was important for growth of human colon cancer cell lines in vitro and in vivo . Mechanistically, MAZ is critical in the regulation of oncogenic STAT3 signaling. MAZ expressing mice have enhanced STAT3 activation in the acute response to colitis. Moreover, MAZ was essential for cytokine and bacteria-induced STAT3 signaling in colon cancer cells. Furthermore, we show that STAT3 is essential for MAZ-induced colon tumorigenesis using a chemical inhibitor. These data indicate an important functional role for MAZ in the inflammatory progression of colon cancer through regulation of STAT3 signaling and suggest MAZ is a potential therapeutic target to dampen STAT3 signaling in colon cancer.
... In spite of being interesting, these results warrant further verification of the nature and role of these proteins in transcription of VEGF promoter. However, evidence shows that some splice variants of SAF protein may act as repressors of VEGF transcription [45]. These findings together suggest a role of metformin treatment in the modulation of the levels as well as VEGF-binding affinity of KLF-4 and SAF-1/Sp1 complexes in the ER+ MCF-7 breast cancer cells. ...
