scholarly journals Analysis of the Arabidopsis organellar rhomboid At1g74140 transcript population uncovered splicing patterns different from its close relative At1g74130

F1000Research ◽  
2019 ◽  
Vol 8 ◽  
pp. 1925
Author(s):  
Kenton Ko ◽  
Jeremy Guenther ◽  
Nicholas Ostan ◽  
Joshua Powles
Keyword(s):  
Gene Expression ◽  
Alternative Splicing ◽  
Splice Variants ◽  
The Other ◽  
Close Relative ◽  
Rt Pcr ◽  
Splicing Pattern ◽  
High Level ◽  
Splicing Patterns ◽  
Alternative Splice Variants

Background: Four distinct rhomboid genes appear to function in Arabidopsis plastids, two “active” types from the secretases and presenilin-like associated rhomboid-like (PARL) categories (At1g25290 and At5g25752) and two “inactive” rhomboid forms (At1g74130 and At1g74140).  The number of working rhomboids is further increased by alternative splicing, two reported for At1g25290 and three for At1g74130.  Since At1g25290 and At1g74130 exist as alternative splice variants, it would be necessary to assess the splicing patterns of the other two plastid rhomboid genes, At5g25752 and At1g74140, before studying the Arabidopsis plastid rhomboid system as a whole.   Methods: This study thus specifically focused on an analysis of the At1g74140 transcript population using various RT-PCR strategies.   Results: The exon mapping results indicate splicing patterns different from the close relative At1g74130, despite similarity between the exonic sequences.  The splicing patterns indicate a high level of sequence “discontinuity” in the At1g74140 transcript population with a significant portion of the discontinuity being generated by two regions of the gene.   Conclusion: The overall discontinuous splicing pattern of At1g74140 may be reflective of its mode of involvement in activities like controlling gene expression.

scholarly journals Regulation of Multiple Core Spliceosomal Proteins by Alternative Splicing-Coupled Nonsense-Mediated mRNA Decay

2008 ◽  
Vol 28 (13) ◽  
pp. 4320-4330 ◽  
Author(s):  
Arneet L. Saltzman ◽  
Yoon Ki Kim ◽  
Qun Pan ◽  
Matthew M. Fagnani ◽  
Lynne E. Maquat ◽  
...  
Keyword(s):  
Gene Expression ◽  
Alternative Splicing ◽  
Mrna Decay ◽  
Splice Variants ◽  
Cellular Functions ◽  
Regulate Gene Expression ◽  
Premature Termination Codons ◽  
Microarray Profiling ◽  
Nonsense Mediated Mrna Decay ◽  
Regulate Gene

ABSTRACT Alternative splicing (AS) can regulate gene expression by introducing premature termination codons (PTCs) into spliced mRNA that subsequently elicit transcript degradation by the nonsense-mediated mRNA decay (NMD) pathway. However, the range of cellular functions controlled by this process and the factors required are poorly understood. By quantitative AS microarray profiling, we find that there are significant overlaps among the sets of PTC-introducing AS events affected by individual knockdown of the three core human NMD factors, Up-Frameshift 1 (UPF1), UPF2, and UPF3X/B. However, the levels of some PTC-containing splice variants are less or not detectably affected by the knockdown of UPF2 and/or UPF3X, compared with the knockdown of UPF1. The intron sequences flanking the affected alternative exons are often highly conserved, suggesting important regulatory roles for these AS events. The corresponding genes represent diverse cellular functions, and surprisingly, many encode core spliceosomal proteins and assembly factors. We further show that conserved, PTC-introducing AS events are enriched in genes that encode core spliceosomal proteins. Where tested, altering the expression levels of these core spliceosomal components affects the regulation of PTC-containing splice variants from the corresponding genes. Together, our results show that AS-coupled NMD can have different UPF factor requirements and is likely to regulate many general components of the spliceosome. The results further implicate general spliceosomal components in AS regulation.

Characterization of three alternative splice variants associated with the Arabidopsis rhomboid protein gene At1g74130

Botany ◽  
2013 ◽  
Vol 91 (12) ◽  
pp. 840-849 ◽  
Author(s):  
Joshua Powles ◽  
Katharine Sedivy-Haley ◽  
Eric Chapman ◽  
Kenton Ko
Keyword(s):  
Alternative Splicing ◽  
Alternative Splice ◽  
Splice Variant ◽  
Serine Proteases ◽  
Transmembrane Domain ◽  
Splice Variants ◽  
Genes Encoding ◽  
Different Characteristics ◽  
Alternative Splice Variants

Rhomboid serine proteases are grouped into three main types — secretases, presenilin-like associated rhomboid-like (PARL) proteases, and “inactive” rhomboid proteins. Although the three rhomboid groups are distinct, the different types are likely to operate within the same cell or compartment, such as observed in the plastids of Arabidopsis. There are four distinct plastid rhomboid genes at play in Arabidopsis plastids, two for active types (At1g25290 and At5g25752) and two for inactive forms (At1g74130 and At1g74140). The number of working plastid rhomboids is further increased by alternative splicing, as reported for At1g25290. To understand how the plastid rhomboid system works, it is necessary to identify all rhomboid forms in play. To this end, this study was designed to examine the alternative splicing activities of At1g74130, one of the two genes encoding proteolytically “inactive” plastid rhomboids. The exon mapping and DNA sequencing results obtained here indicate the presence of three prominent alternative splice variants in the At1g74130 transcript population. The dominant splice variant, L, encodes the full-length protein. The other two splice variants, M and S, produce proteins lacking sections from the carboxyl transmembrane domain region. The splice variants M and S appear to be at levels with functional potential and appear to adjust relative to each other during development and in response to changes in the level of Tic40, a component of the plastid translocon. The splice variant proteins themselves exhibit different characteristics with respect to rhomboid protein–substrate interactions. These differences were observed in bacterial co-expression pull-down assays and in yeast mitochondrial studies. When considered together, the data suggest that the alternative splicing of At1g74130 bears functional significance in Arabidopsis and is likely to be part of a mechanism for diversifying plastid rhomboid function.

scholarly journals Cellular stressors may alter islet hormone cell proportions by moderation of alternative splicing patterns

2019 ◽  
Vol 28 (16) ◽  
pp. 2763-2774 ◽  
Author(s):  
Nicola Jeffery ◽  
Sarah Richardson ◽  
David Chambers ◽  
Noel G Morgan ◽  
Lorna W Harries
Keyword(s):  
Gene Expression ◽  
Alternative Splicing ◽  
Kinase Inhibitor ◽  
Ex Vivo ◽  
Regulation Of Gene Expression ◽  
Splicing Factor ◽  
Splicing Regulation ◽  
Messenger Ribonucleic Acid ◽  
Splicing Patterns ◽  
Cellular Stressors

Abstract Changes to islet cell identity in response to type 2 diabetes (T2D) have been reported in rodent models, but are less well characterized in humans. We assessed the effects of aspects of the diabetic microenvironment on hormone staining, total gene expression, splicing regulation and the alternative splicing patterns of key genes in EndoC-βH1 human beta cells. Genes encoding islet hormones [somatostatin (SST), insulin (INS), Glucagon (GCG)], differentiation markers [Forkhead box O1 (FOXO1), Paired box 6, SRY box 9, NK6 Homeobox 1, NK6 Homeobox 2] and cell stress markers (DNA damage inducible transcript 3, FOXO1) were dysregulated in stressed EndoC-βH1 cells, as were some serine arginine rich splicing factor splicing activator and heterogeneous ribonucleoprotein particle inhibitor genes. Whole transcriptome analysis of primary T2D islets and matched controls demonstrated dysregulated splicing for ~25% of splicing events, of which genes themselves involved in messenger ribonucleic acid processing and regulation of gene expression comprised the largest group. Approximately 5% of EndoC-βH1 cells exposed to these factors gained SST positivity in vitro. An increased area of SST staining was also observed ex vivo in pancreas sections recovered at autopsy from donors with type 1 diabetes (T1D) or T2D (9.3% for T1D and 3% for T2D, respectively compared with 1% in controls). Removal of the stressful stimulus or treatment with the AKT Serine/Threonine kinase inhibitor SH-6 restored splicing factor expression and reversed both hormone staining effects and patterns of gene expression. This suggests that reversible changes in hormone expression may occur during exposure to diabetomimetic cellular stressors, which may be mediated by changes in splicing regulation.

scholarly journals Alternative Splicing During the Chlamydomonasreinhardtii Cell Cycle

2020 ◽  
Vol 10 (10) ◽  
pp. 3797-3810
Author(s):  
Manishi Pandey ◽  
Gary D. Stormo ◽  
Susan K. Dutcher
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Alternative Splicing ◽  
Chlamydomonas Reinhardtii ◽  
Intron Retention ◽  
Exon Skipping ◽  
Dark Phase ◽  
Periodic Patterns ◽  
Genome Wide ◽  
Splicing Pattern

Genome-wide analysis of transcriptome data in Chlamydomonas reinhardtii shows periodic patterns in gene expression levels when cultures are grown under alternating light and dark cycles so that G1 of the cell cycle occurs in the light phase and S/M/G0 occurs during the dark phase. However, alternative splicing, a process that enables a greater protein diversity from a limited set of genes, remains largely unexplored by previous transcriptome based studies in C. reinhardtii. In this study, we used existing longitudinal RNA-seq data obtained during the light-dark cycle to investigate the changes in the alternative splicing pattern and found that 3277 genes (19.75% of 17,746 genes) undergo alternative splicing. These splicing events include Alternative 5′ (Alt 5′), Alternative 3′ (Alt 3′) and Exon skipping (ES) events that are referred as alternative site selection (ASS) events and Intron retention (IR) events. By clustering analysis, we identified a subset of events (26 ASS events and 10 IR events) that show periodic changes in the splicing pattern during the cell cycle. About two-thirds of these 36 genes either introduce a pre-termination codon (PTC) or introduce insertions or deletions into functional domains of the proteins, which implicate splicing in altering gene function. These findings suggest that alternative splicing is also regulated during the Chlamydomonas cell cycle, although not as extensively as changes in gene expression. The longitudinal changes in the alternative splicing pattern during the cell cycle captured by this study provides an important resource to investigate alternative splicing in genes of interest during the cell cycle in Chlamydomonas reinhardtii and other eukaryotes.

scholarly journals Ovarian steroids regulate 24p3 expression in mouse uterus during the natural estrous cycle and the preimplantation period

1999 ◽  
Vol 162 (1) ◽  
pp. 11-19 ◽  
Author(s):  
HL Huang ◽  
ST Chu ◽  
YH Chen
Keyword(s):  
Gene Expression ◽  
Estrous Cycle ◽  
Immunohistochemical Localization ◽  
The Other ◽  
Undetectable Level ◽  
Mouse Uterus ◽  
Ovarian Steroids ◽  
Vaginal Plug ◽  
High Level

We examined 24p3 expression in the mouse uterus at various stages of the natural estrous cycle and during the preimplantation period. The level of 24p3 mRNA appeared intensively in proestrus and estrus, then declined sharply from metestrus to diestrus. Consistent with this observation, 24p3 protein was abundant in proestrus, decreased from estrus to metestrus and declined to a very low level in diestrus. The uterine 24p3 expression closely overlapped with the estradiol (E2) surge in proestrus and estrus but it was suppressed when progesterone (P4) rose to a high level during the reproductive cycle. Neither the protein nor its message was detected in the uteri of immature mice or ovariectomized adult animals. While an injection of P4 to these animals was unable to initiate uterine 24p3 expression, administration of estrogenic steroids to these animals markedly stimulated the gene expression. Treatment of these animals with E2 together with P4, on the other hand, did not stimulate the gene expression. In pregnant animals (day 1 (D1)=day of vaginal plug), 24p3 mRNA remained at a high level on D1 and D2 but dropped to an almost undetectable level on D3 and D4. This was accompanied by a decrease in 24p3 protein from D1 to D2 and a decline in the protein to undetectable levels from D3 to D4. The staining patterns of both the immunohistochemical localization of 24p3 protein and in situ hybridization for the detection of 24p3 mRNA in the uterine sections showed that 24p3 expression took place mainly in the luminal and glandular epithelial cells of the endometrium. This together with our previous observation that 24p3 protein is found in uterine luminal fluid indicates that the protein is secreted primarily from these cells to their respective luminal surfaces during proestrus and estrus.

Identification of Alternative Splicing Changes in Erythroid Differentiation Using Human Exon Arrays.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1707-1707
Author(s):  
Miki L. Yamamoto ◽  
Jeong-Ah Kang ◽  
Josh A. Arribere ◽  
Amittha Wickrema ◽  
John G. Conboy
Keyword(s):  
Gene Expression ◽  
Alternative Splicing ◽  
Erythroid Differentiation ◽  
Transcriptional Level ◽  
Rt Pcr ◽  
Erythroid Progenitor ◽  
Array Data ◽  
Exon Arrays ◽  
Human Exon ◽  
A Genome

Abstract Terminal erythroid differentiation is accompanied by extensive structural remodeling as the cell enucleates and eventually assumes the biconcave disk morphology of the mature cell. Previous studies have documented many changes at the transcriptional level essential for erythroid differentiation. Changes in erythroid gene expression also occur at the level of pre-mRNA alternative splicing: the activation of 4.1R (EPB41) exon 16 splicing in late erythroblasts increases 4.1R affinity for spectrin-actin and mechanically strengthens the plasma membrane. We hypothesize that analogous changes in alternative splicing affect the structure and function of other erythroid proteins. To identify additional alternative splicing switches in erythroid genes, a genome-wide exon expression analysis was carried out using the new Affymetrix Human Exon 1.0 ST Array. Unlike traditional gene expression microarrays, this array has single exon resolution and can detect changes in expression due to alternative splicing. Samples for array analyses were prepared from RNA of human erythroid progenitor cells grown in culture for 7, 10, and 14 days, corresponding to basophilic, polychromatic, and orthochromatic stages. Analysis of this exon array data confirmed that 4.1R exon 16 splicing was activated in day 14 cells, and that a known inhibitor of exon 16 splicing, hnRNP A1, was down-regulated in coordination with the 4.1R splicing switch. As another positive control, we confirmed in array data the expression of a known erythroid-specific 3′ end in beta-spectrin mRNA in all three time points of erythroblasts, while array data from muscle tissue showed expression of only the non-erythroid 3′ end of beta-spectrin. Array data is now being analyzed to identify new cases of alternative splicing during erythropoiesis, and confirmation of several candidate splicing switches by RT-PCR and quantitative PCR is under way. A number of genes, including PIK3R1, SLC12A6, and TNPO2, show changes in alternative 5′ first exon usage during late erythropoiesis. A splicing change involving an internal cassette exon in MBNL2, which encodes a splicing regulator, was identified by array data and confirmed by RT-PCR. In addition, overall gene expression analyses confirm up-regulation of known genes expressed during erythroid differentiation, including Band 3, GLUT1, ALAS2, and BCL2L1. This preliminary analysis demonstrates the application of exon arrays toward the identification of splicing switches that occur during differentiation of human erythroblasts. Further validation of putative alternative splicing events is in progress, and investigation of the regulation of the validated events and the physiological implications of the predicted changes in the proteins will be pursued in the future.

scholarly journals Ontogeny of erythropoietin gene expression in the sheep fetus: effect of dexamethasone at 60 days of gestation

Blood ◽  
1994 ◽  
Vol 84 (2) ◽  
pp. 460-466
Author(s):  
GB Lim ◽  
K Jeyaseelan ◽  
EM Wintour
Keyword(s):  
Gene Expression ◽  
Mrna Levels ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Fetal Plasma ◽  
Liver And Kidney ◽  
Polymerase Chain ◽  
High Level ◽  
A Site ◽  
Sheep Fetus

We have used competitive reverse transcription and polymerase chain reaction (RT/PCR) to compare the levels of erythropoietin (Epo) mRNA in the liver and kidneys of the sheep fetus at 60, 80, 100, 130, and 140 days of gestation (term = 145 to 150 days). The effect of dexamethasone infusion in the ewe on Epo gene expression in the 60-day fetus was also investigated. Epo mRNA levels were highest at 60 days of gestation, the earliest age studied, in both liver and kidney. In the liver, Epo mRNA expression declined as gestation proceeded. Kidney Epo mRNA was maintained at a high level until 100 days of gestation, declining significantly in the 130-day fetus (P < .01). Treatment of ewes carrying 60-day fetuses with 0.76 mg/h dexamethasone for 48 hours resulted in a significant decrease in fetal plasma Epo values and Epo mRNA levels in both the liver and kidney. In the dexamethasone-treated fetuses, Epo mRNA in the liver was 52% of control values (P < .05), and in the kidney, 33% of control (P < .001). The results suggest that the kidney may play a more important role as a site of Epo synthesis in the early gestation sheep fetus than previously thought. Glucocorticoids may have a role in the regulation of Epo gene expression.

Identification of New Megakaryocytic Subpopulations in Mouse Bone Marrow.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2265-2265
Author(s):  
Shinji Sogo ◽  
Kuniko Matsumura-Takeda ◽  
Yoshimasa Isakari ◽  
Yasuo Harada ◽  
Kinue Nishioka ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Gene Expression Profiling ◽  
Expression Profiling ◽  
Molecular Mechanisms ◽  
Present Finding ◽  
The Other ◽  
Mouse Bone Marrow ◽  
Rt Pcr ◽  
Kit Gene

Abstract Platelets (PLT) are produced from megakaryocytes (Mks) via proplatelet formation (PPF). However, the molecular mechanisms from Mks to PPF are not clearly elucidated, because the maturational steps of the Mks in bone marrow (BM) are not analyzed in detail. Until now, mouse Mks have been only isolated as acetylcholinesterase (AchE) positive cells and they are understood as well maturated population. In this study, we found the presence of different megakaryocytic subpopulations in BM by flowcytometry. To isolate the Mks, first we depleted lineage marker (CD4, CD8a, CD11b, B220, CD71, CD90, TER119, Gr-1, F4/80, 7/4) positive cells from BM cells of BALB/c mice. The analysis of the expression-pattern of CD41, CD45 and CD61 in the lineage negative (Lin−) cells showed the presence of two types of megakaryocytic subpopulations. By sorting, they were identified as Lin−CD41+/45+/61+ cells (AchE negative) and Lin−CD41++/45+/61++ cells (partially AchE positive), respectively. To assess the maturational stages of the subpopulations, each population was cultured with 10ng/mL of TPO followed by counting of PPF and PLT production. Both PPF and PLT production were observed in Lin−CD41+/45+/61+ cells later than those in Lin−CD41++/45+/61++ cells. On the other hand, CFU-Mk was scarcely detected in each subpopulation. The results indicate that both populations are the committed megakaryocytes and Lin−CD41+/45+/61+ cells are more immature population than Lin−CD41++/45+/61++ cells. Then to characterize these subpopulations in detail, gene expression profiling was performed against four-megakaryocytic lineage-populations, Lin−CD41−Thy1lowc-kit+ cells as stem/progenitor, Lin−CD41+/45+/61+ cells, Lin−CD41++/45+/61++ cells and PLT using GeneChipU74 or RT-PCR. These analyses revealed that many PLT-specific genes including gpIb/IX, P-selectin, thrombin-R and ADP-R were already expressed on Lin−CD41+/45+/61+ cells but less than Lin−CD41++/45+/61++ cells. Especially, beta-1 tubulin that is necessary for PPF was only expressed on Lin−CD41++/45+/61++ cells. On the contrary, the expression of c-kit gene was gradually decreasing from stem/progenitor fraction to PLT. In conclusion, we succeeded in the isolation of new subpopulations distinguishable between immature Mks and more matured Mks beginning to prepare PLT. The present finding can contribute to elucidate the molecular mechanisms during terminal maturation.

scholarly journals Origin, Splicing, and Expression of Rodent Amelogenin Exon 8

2006 ◽  
Vol 85 (10) ◽  
pp. 894-899 ◽  
Author(s):  
J.D. Bartlett ◽  
R. L. Ball ◽  
T. Kawai ◽  
C.E. Tye ◽  
M. Tsuchiya ◽  
...  
Keyword(s):  
Alternative Splicing ◽  
Splice Site ◽  
Site Selection ◽  
Proteolytic Processing ◽  
Rt Pcr ◽  
Splice Site Selection ◽  
Rna Transcripts ◽  
Splicing Pattern ◽  
Alternatively Spliced ◽  
Gene Structures

Amelogenin RNA transcripts undergo extensive alternative splicing, and MMP-20 processes the isoforms following their secretion. Since amelogenins have been ascribed cell-signaling activities, we asked if a lack of proteolytic processing by MMP-20 affects amelogenin signaling and consequently alters amelogenin splice site selection. RT-PCR analyses of amelogenin mRNA between control and Mmp20− /−mice revealed no differences in the splicing pattern. We characterized 3 previously unidentified amelogenin alternatively spliced transcripts and demonstrated that exon-8-encoded amelogenin isoforms are processed by MMP-20. Transcripts with exon 8 were expressed approximately five-fold less than those with exon 7. Analyses of the mouse and rat amelogenin gene structures confirmed that exon 8 arose in a duplication of exons 4 through 5, with translocation of the copy downstream of exon 7. No downstream genomic sequences homologous to exons 4–5 were present in the bovine or human amelogenin genes, suggesting that this translocation occurred only in rodents.

scholarly journals Multiple splice variants of the pituitary adenylate cyclase-activating polypeptide type 1 receptor detected by RT-PCR in single rat pituitary cells

1998 ◽  
Vol 21 (2) ◽  
pp. 109-120 ◽  
Author(s):  
L Bresson-Bepoldin ◽  
MC Jacquot ◽  
W Schlegel ◽  
SR Rawlings
Keyword(s):  
Alternative Splicing ◽  
Adenylate Cyclase ◽  
Single Cell ◽  
Splice Variant ◽  
Cell Biology ◽  
Splice Variants ◽  
Pituitary Cells ◽  
Rt Pcr ◽  
Pituitary Adenylate Cyclase

Alternative splicing of the rat type 1 pituitary adenylate cyclase-activating polypeptide (PACAP) receptor (PVR1) produces variants that couple either to both adenylyl cyclase (AC) and phospholipase C (PLC) (PVR1 short, PVR1 hop, PVR1 hiphop), or to AC alone (PVR1 hip). We have previously shown that populations of clonal alphaT3-1 gonadotrophs express PVR1 hop and PVR1 short mRNAs, whereas clonal GH4C1 somatotrophs do not. Here we have used the single cell RT-PCR technique to investigate whether normal rat gonadotrophs and somatotrophs express PVR1 mRNA, whether a single cell co-expresses multiple splice variant forms, and whether differential PVR1 mRNA expression correlates with differences in PACAP-stimulated Ca2+ signalling. We found that individual rat gonadotrophs expressed mRNA either for PVR1 hop, for PVR1 short, or co-expressed the two forms. Although we found no differences between the splice variant(s) expressed and the characteristics of PACAP-stimulated Ca2+ responses, the expression of PVR1 mRNA is consistent with the known PACAP stimulation of the PLC system in gonadotrophs. Individual rat somatotrophs also expressed PVR1 hop or PVR1 short (but not PVR1 hip) mRNAs although these forms were never co-expressed. The expression of PVR1 mRNA in somatotrophs can explain in part the activation by PACAP of the AC system in such cells. In conclusion, the single cell RT-PCR technique was used to demonstrate expression of multiple PVR1 splice variants in single identified pituitary cells. These findings open up important questions on the role of alternative splicing in cell biology.

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