Alternative splicing variability: exactly how similar are two identical cells?

Modification of proteins by small ubiquitin-like modifier (SUMO) is emerging as an important control of transcription and RNA processing. The human factor TCERG1 (also known as CA150) participates in transcriptional elongation and alternative splicing of pre-mRNAs. Here, we report that SUMO family proteins modify TCERG1. Furthermore, TCERG1 binds to the E2 SUMO-conjugating enzyme Ubc9. Two lysines (Lys-503 and Lys-608) of TCERG1 are the major sumoylation sites. Sumoylation does not affect localization of TCERG1 to the splicing factor-rich nuclear speckles or the alternative splicing function of TCERG1. However, mutation of the SUMO acceptor lysine residues enhanced TCERG1 transcriptional activity, indicating that SUMO modification negatively regulates TCERG1 transcriptional activity. These results reveal a regulatory role for sumoylation in controlling the activity of a transcription factor that modulates RNA polymerase II elongation and mRNA alternative processing, which are discriminated differently by this post-translational modification.

Here, we report the identification of the RNA binding motif protein RBM15B/OTT3 as a new CDK11(p110) binding partner that alters the effects of CDK11 on splicing. RBM15B was initially identified as a binding partner of the Epstein-Barr virus mRNA export factor and, more recently, as a cofactor of the nuclear export receptor NXF1. In this study, we found that RBM15B co-elutes with CDK11(p110), cyclin L2α, and serine-arginine (SR) proteins, including SF2/ASF, in a large nuclear complex of ∼1-MDa molecular mass following size exclusion chromatography. Using co-immunoprecipitation experiments and in vitro pulldown assays, we mapped two distinct domains of RBM15B that are essential for its direct interaction with the N-terminal extension of CDK11(p110), cyclin L2α, and SR proteins such as 9G8 and SF2/ASF. Finally, we established that RBM15B is a functional competitor of the SR proteins SF2/ASF and 9G8, inhibits formation of the functional spliceosomal E complex, and antagonizes the positive effect of the CDK11(p110)-cyclin L2α complex on splicing both in vitro and in vivo.

Human DNA topoisomerase I plays a dual role in transcription, by controlling DNA supercoiling and by acting as a specific kinase for the SR-protein family of splicing factors. The two activities are mutually exclusive, but the identity of the molecular switch is unknown. Here we identify poly(ADP-ribose) as a physiological regulator of the two topoisomerase I functions. We found that, in the presence of both DNA and the alternative splicing factor/splicing factor 2 (ASF/SF2, a prototypical SR-protein), poly(ADP-ribose) affected topoisomerase I substrate selection and gradually shifted enzyme activity from protein phosphorylation to DNA cleavage. A likely mechanistic explanation was offered by the discovery that poly(ADP-ribose) forms a high affinity complex with ASF/SF2 thereby leaving topoisomerase I available for directing its action onto DNA. We identified two functionally important domains, RRM1 and RS, as specific poly(ADP-ribose) binding targets. Two independent lines of evidence emphasize the potential biological relevance of our findings: (i) in HeLa nuclear extracts, ASF/SF2, but not histone, phosphorylation was inhibited by poly(ADP-ribose); (ii) an in silico study based on gene expression profiling data revealed an increased incidence of alternative splicing within a subset of inflammatory response genes that are dysregulated in cells lacking a functional poly(ADP-ribose) polymerase-1. We propose that poly(ADP-ribose) targeting of topoisomerase I and ASF/SF2 functions may participate in the regulation of gene expression.

Discoidin domain receptors (DDRs) DDR1 and DDR2 are receptor tyrosine kinases with the unique ability among receptor tyrosine kinases to respond to collagen. Several signaling molecules have been implicated in DDR signaling, including Shp-2, Src, and MAPK pathways, but a detailed understanding of these pathways and their transcriptional targets is still lacking. Similarly, the regulation of the expression of DDRs is poorly characterized with only a few inflammatory mediators, such as lipopolysaccharide and interleukin-1β identified as playing a role in DDR1 expression. DDRs have been reported to induce the expression of various genes including matrix metalloproteinases and bone morphogenetic proteins, but the regulatory mechanisms underlying DDR-induced gene expression remain to be determined. The aim of the present work was to elucidate the molecular mechanisms implicated in the expression of DDRs and to identify DDR-induced signaling pathways and target genes. Our data show that collagen I induces the expression of DDR1 in a dose- and time-dependent manner in primary human lung fibroblasts. Furthermore, activation of DDR2, JAK2, and ERK1/2 MAPK signaling pathways was essential for collagen I-induced DDR1 and matrix metalloproteinase 10 expression. Finally, inhibition of the ERK1/2 pathway abrogated DDR1 expression by blocking the recruitment of the transcription factor polyoma enhancer A-binding protein 3 to the DDR1 promoter. Our data provide new insights into the molecular mechanisms of collagen I-induced DDR1 expression and demonstrate an important role for ERK1/2 activation and the recruitment of polyoma enhancer-A binding protein 3 to the DDR1 promoter.

We report the existence of eight different interleukin-15 receptor alpha-chain (IL-15Ralpha) transcripts resulting from exon-splicing mechanisms within the IL-15Ralpha gene. Two main classes of transcripts can be distinguished that do or do not (Delta2 isoforms) contain the exon 2-coding sequence. Both classes were expressed in numerous cell lines and tissues (including peripheral blood lymphocytes) at comparable levels and could be transcribed in COS-7 cells, and the proteins were expressed at the cell surface. Both receptor forms displayed numerous glycosylation states, reflecting differential usage of a single N-glycosylation site as well as extensive O-glycosylations. Whereas IL-15Ralpha bound IL-15 with high affinity, Delta2IL-15Ralpha was unable to bind IL-15, thus revealing the indispensable role of the exon 2-encoded domain in cytokine binding. A large proportion of IL-15Ralpha was expressed at the nuclear membrane with some intranuclear localization, supporting a potential direct action of the IL-15.IL-15Ralpha complex at the nuclear level. In sharp contrast, Delta2IL-15Ralpha was found only in the non-nuclear membrane compartments, indicating that the exon 2-encoded domain (which is shown to contain a potential nuclear localization signal) plays an important role in receptor post-translational routing. Together, our data indicate that exon 2 splicing of human IL-15Ralpha is a natural process that might play regulatory roles at different levels.

In this study, the first nestin isoform, Nes-S, was identified in neurons of dorsal root ganglia (DRG) of adult rats. Nes-S cannot form filaments by itself in cytoplasmic intermediate filament-free SW13 cells. Instead, it co-assembles into filaments with vimentin when transfected into vimentin(+) SW13 cells, and with peripherin and neurofilament proteins when transfected into N2a cells. In primary DRG neurons, endogenous Nes-S co-assembles with peripherin and neurofilament proteins. The expression of Nes-S first appears in DRG at postnatal day 5 and persists to adulthood. Among the adult tissues we examined, the expression of Nes-S is restricted to the sensory and motor neurons. Finally, exogenous Nes-S enhances viability when transfected into N2a cells, and knockdown of endogenous Nes-S impairs the survival of DRG neurons in primary cultures. Taken together, Nes-S is a new neuronal intermediate filament protein that exerts a cytoprotective function in mature sensory and motor neurons.

RNA Splicing: A Novel Pathogen Effector Target

LKB1 is an upstream activating kinase for the AMP-activated protein kinase (AMPK) and at least 12 other AMPK-related kinases. LKB1 therefore acts as a master kinase regulating the activity of a wide range of downstream kinases, which themselves have diverse physiological roles. Here we identify a second form of LKB1 generated by alternative splicing of the LKB1 gene. The two LKB1 proteins have different C-terminal sequences generating a 50-kDa form (termed LKB1L) and a 48-kDa form (LKB1S). LKB1L is widely expressed in mouse tissues, whereas LKB1S has a restricted tissue distribution with predominant expression in the testis. LKB1S, like LKB1L, forms a complex with MO25 and STRAD, and phosphorylates and activates AMPK both in vitro and in intact cells. A phosphorylation site (serine 431 in mouse) and a farnesylation site (cysteine 433 in mouse) within LKB1L are not conserved in LKB1S raising the possibility that these sites might be involved in differential regulation and/or localization of the two forms of LKB1. However, we show that phosphorylation of serine 431 has no effect on LKB1L activity and that both LKB1L and LKB1S have similar patterns of subcellular localization. These results indicate that the physiological significance of the different forms of LKB1 is not related directly to differences in the C-terminal sequences but may be due to their differential patterns of tissue distribution.

Proteolytic Cleavage of the CD44 Adhesion Molecule in Multiple Human Tumors

Many eukaryotic transcription factors are bimodal in their regulatory properties and can both repress and activate expression of their target genes. These divergent transcriptional properties are conferred through recruitment of auxiliary proteins, denoted coactivators and corepressors. Repression plays a particularly critical role in the functions of the nuclear receptors, a large family of ligand-regulated transcription factors involved in metazoan development, differentiation, reproduction, and homeostasis. The SMRT corepressor interacts directly with nuclear receptors and serves, in turn, as a platform for the assembly of a larger corepressor complex. We report here that SMRT is expressed in cells by alternative mRNA splicing to yield two distinct variants or isoforms. We designate these isoforms SMRTalpha and SMRTtau and demonstrate that these isoforms have significantly different affinities for different nuclear receptors. These isoforms are evolutionarily conserved and are expressed in a tissue-specific manner. Our results suggest that differential mRNA splicing serves to customize corepressor function in different cells, allowing the transcriptional properties of nuclear receptors to be adapted to different contexts.

The myeloid C-type lectin dendritic cell-specific ICAM3-grabbing non-integrin (DC-SIGN, CD209) recognizes oligosaccharide ligands on clinically relevant pathogens (HIV, Mycobacterium, and Aspergillus). Alternative splicing and genomic polymorphism generate DC-SIGN mRNA variants, which have been detected at sites of pathogen entrance and transmission. We present evidence that DC-SIGN neck variants are expressed on dendritic and myeloid cells at the RNA and protein levels. Structural analysis revealed that multimerization of DC-SIGN within a cellular context depends on the lectin domain and the number and arrangement of the repeats within the neck region, whose glycosylation negatively affects oligomer formation. Naturally occurring DC-SIGN neck variants differ in multimerization competence in the cell membrane, exhibit altered sugar binding ability, and retain pathogen-interacting capacity, implying that pathogen-induced cluster formation predominates over the basal multimerization capability. Analysis of DC-SIGN neck polymorphisms indicated that the number of allelic variants is higher than previously thought and that multimerization of the prototypic molecule is modulated in the presence of allelic variants with a different neck structure. Our results demonstrate that the presence of allelic variants or a high level of expression of neck domain splicing isoforms might influence the presence and stability of DC-SIGN multimers on the cell surface, thus providing a molecular explanation for the correlation between DC-SIGN polymorphisms and altered susceptibility to HIV-1 and other pathogens.

It is now well established that eukaryotic cells respond to their changing milieu through coordinate transcriptional and post-transcriptional mechanisms. Post-transcriptional mechanisms include those that tune the expression of a battery of genes encoding proteins of diverse function, if not mRNAs of diverse structure, via targeted mRNA decay. As recently as 10 years ago, a change in mRNA abundance that was less than five- to 10-fold was deemed by many to be physiologically insignificant. However, an appreciation for the importance of small changes has grown as a consequence of new methodologies that are sufficiently sensitive and sophisticated to quantitate small and concomitant changes to the abundance of groups of mRNAs. Only relatively recently—since the inception of technological advances in transcriptome deep-sequencing and RT-coupled to real-time PCR that enable scientists to reliably measure changes in RNA abundance that are less than 1.5-fold and that typify groups of RNAs—has the existence of a truly remarkable and previously inconceivable network of post-transcriptional processes been uncovered. While this network is far from completely established, it has become clear that it consists of responses that individually do not necessarily promote an appropriate cellular response but together, and in conjunction with transcriptional and other regulated pathways, most certainly do. The idea that the sum of small changes can have significant consequences is relevant to a broad range of cellular processes, including epigenomic modifications to DNA. Nevertheless, evidence presented here will be limited to mRNA decay. Furthermore, while the focus will be on nonsense-mediated mRNA decay (NMD), small changes in the half-lives of groups of mRNAs as part of a cellular response to changes in cellular environment also characterize other mRNA decay pathways, as exemplified by many in studies of microRNA-mediated mRNA decay and my lab and the lab of Yoon Ki Kim on Staufen-mediated mRNA decay. NMD: Easy does it NMD is largely a quality-control pathway that degrades mRNAs that prematurely terminate translation so as to obviate the production of truncated proteins that are potentially toxic to cells. These mRNAs are often the result of mistakes made during gene expression, and in humans they include an estimated one-third of alternatively spliced mRNAs as well as an estimated two-thirds of mRNAs that derive from genes that undergo somatic-cell rearrangement and hyper-mutation. Furthermore, NMD targets are responsible for approximately one-third of inherited diseases because they harbor a frameshift or nonsense mutation that generates a premature termination codon and precludes the production of functional protein. Pertinent to this piece, NMD additionally degrades ∼10% of normal mRNAs. These mRNAs, which derive from splicing (since unspliced mRNAs are generally not NMD targets), are heterogenous in structure, encode proteins of diverse functions, and are NMD targets for one of a variety of reasons: for example, they harbor an upstream open translational reading frame, an 3′-untranslated region (3′UTR) intron, or an abnormally long 3′UTR, each of which may be the consequence of alternative pre-mRNA splicing or 3′-end formation. Relatively recently, data indicate that NMD is used by cells to fine-tune gene expression in response to environmental stimuli. For example, my lab has shown that during muscle-cell differentiation in not only humans but also rodents, the efficiency of NMD is downregulated approximately twofold (as determined using β-globin-based NMD reporters to compare the efficiency of NMD in undifferentiated myoblasts vs. differentiated myotubes) while the efficiency of a competing pathway, Staufen-mediated mRNA decay (SMD), is upregulated approximately twofold (as determined using luciferase-based SMD reporters to compare the efficiency of SMD in undifferentiated myoblasts vs. differentiated myotubes). These small and opposing changes to the efficiencies of SMD and NMD during myogenesis offer means to downregulate a pool of mRNAs that includes some whose protein products maintain myoblasts in an undifferentiated state as well as upregulate a pool of mRNAs that includes some whose protein products encode pro-myogenic factors. Lawrence Gardner's lab has found that the efficiency of NMD is also inhibited ∼2.5-fold during hypoxia (as determined using β-globin-based NMD reporters in a comparison of prostate cancer PC3 cells grown as monolayers to PC3 cells grown as three-dimensional tumors) so as to promote tumorigenesis. This seems to have the effect of stabilizing NMD substrates that promote the response to stress (including genes that are responsive to the unfolded-protein response), allowing cells to adapt to their newly established environmental conditions. During the differentiation of neural stem cells, the group of Miles Wilkinson has demonstrated that NMD is downregulated in part by the expression of a neural-specific microRNA, miR-128, that partially degrades mRNA encoding the key NMD factor UPF1. As a consequence, the efficiency of NMD is abrogated approximately twofold (as measured using a luciferase-activity reporter assay during 4 d of PC12 pheochromocytoma-cell neurogenesis) so as to upregulate the abundance of mRNAs that include those encoding proteins promoting post-mitotic neuronal differentiation. As a final but certainly not the last example, Max Popp in my lab has shown that a sufficient level of DNA damage induced by commonly used chemotherapeutics, such as doxorubicin, inhibits NMD approximately twofold by triggering the caspase-mediated cleavage of some but not all of UPF1 protein, thereby upregulating the half-lives of mRNAs encoding proteins that mediate programmed cell death. Notably, in each case, the modest inhibition of NMD is necessary but not sufficient for the appropriate cellular response. Max demonstrated this explicitly in the case programmed cell death by recapitulating, in the absence of DNA damage (i.e., in the absence of doxorubicin), the physiologic level of cleaved UPF1 observed in the presence of doxorubicin by co-expressing tobacco etch viral (TEV) protease and UPF1 engineered to contain a TEV protease cleavage site. Data from the group of Ari Helenius has offered a twist to this theme: various plus-stranded RNA viruses may have evolved mechanisms to reduce the degradation of their own transcripts by inhibiting the cellular NMD machinery, and these viruses would be sub-optimally served if their inhibitory mechanism severely debilitated host-cell viability. Furthermore, following an immune response, Jonathan Dinman's lab has found that the low efficiency with which various cytokine receptor-encoding mRNAs are targeted for NMD as a consequence of microRNA-promoted −1 programmed ribosomal frameshifting (measured using dual luciferase reporters of the CCR5 mRNA to be 4.5%–11% depending on cell type) pushes cells towards immune desensitization as a way for individual cells to fine-tune their return to homeostasis. In summary, the effects of post-transcriptional regulatory pathways that have more than one target transcript are often more than the sum of the effects of targeting individual transcripts. This is certainly the case for NMD. Given new technologies able to detect transcript levels that vary by less than twofold, we now realize that cells utilize NMD to only modestly alter the expression of groups of genes. By encoding pools of NMD targets that can respond appropriately to a variety of cellular changes, cells are able to tune their metabolic state with a robust versatility.

Head and neck squamous cell carcinoma (HNSC) is a common malignancy with high mortality and poor prognosis. Alternative splicing (AS) is a transcriptional regulation mechanism that generates multiple transcripts from same genes, and aberrant AS signatures of cancers can be predictive for prognosis. We identified the survival-related AS events and splicing factors (SFs) from the RNA sequencing data and the corresponding clinical information of an HNSC cohort downloaded from The Cancer Genome Atlas (TCGA) and SpliceSeq. The independent prognostic predictors were assessed by Cox proportional regression analysis, and the regulatory network of SFs and AS events was analyzed by Spearman's test and constructed. A total of 4626 survival-related AS events in 3280 genes were identified, and most were protective factors. Among the different types of splicing events, exon skip was the most frequent. The prognostic models were constructed for each type of AS, and the area under the curve of the receiver operating characteristic curve of the combined prognostic model was 0.765, indicating good predictive performance. Finally, a correlation network between SF and AS events was constructed. We identified prognostic predictors based on AS events that stratified HNSC patients into the high- and low-risk groups, and revealed splicing networks that provide insights into the underlying mechanisms.

AMPA Receptor Subunit-Specific Regulation by a Distinct Family of Type II TARPs

Nonsense-mediated mRNA decay (NMD) is an evolutionarily conserved surveillance mechanism across eukaryotes and also regulates the expression of physiological transcripts, thus involved in gene regulation. It essentially ensures recognition and removal of aberrant transcripts. Therefore, the NMD protects the cellular system by restricting the synthesis of truncated proteins, potentially by eliminating the faulty mRNAs. NMD is an evolutionarily conserved surveillance mechanism across eukaryotes and also regulates the expression of physiological transcripts, thus involved in gene regulation as well. Primarily, the NMD machinery scans and differentiates the aberrant and non-aberrant transcripts. A myriad of cellular dysfunctions arise due to production of truncated proteins, so the NMD core proteins, the up-frameshift factors (UPFs) recognizes the faulty mRNAs and further recruits factors resulting in the mRNA degradation. NMD exhibits astounding variability in its ability in regulating cellular mechanisms including both pathological and physiological events. But, the detailed underlying molecular mechanisms in NMD remains blurred and require extensive investigation to gain insights on cellular homeostasis. The complexity in understanding of NMD pathway arises due to the involvement of numerous proteins, molecular interactions and their functioning in different steps of this process. Moreover methods such as alternative splicing generates numerous isoforms of mRNA, so it makes difficulties in understanding the impact of alternative splicing on the efficiency of NMD functioning. Role of NMD in cancer development is very complex. Studies have shown that in some cases cancer cells use NMD pathway as a tool to exploit the NMD mechanism to maintain tumor microenvironment. A greater level of understanding about the intricate mechanism of how tumor used NMD pathway for their benefits, a strategy can be developed for targeting and inhibiting NMD factors involved in pro-tumor activity. There are very little amount of information available about the NMD pathway, how it discriminate mRNAs that are targeted by NMD from those that are not. This review highlights our current understanding of NMD, specifically the regulatory mechanisms and attempts to outline less explored questions that warrant further investigations. Taken as a whole, a detailed molecular understanding of the NMD mechanism could lead to wide-ranging applications for improving cellular homeostasis and paving out strategies in combating pathological disorders leaping forward toward achieving United Nations sustainable development goals (SDG 3: Good health and well-being).