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HMGA1a-U1 snRNP complex formation depends on the HMGA1a binding sequence and adjacent 5 splice site. (Top) Aligned sequences of the short RNAs that contain a single copy of the HMGA1a-binding sequence (18) and adjacent 5 splice site. Asterisks indicate mutated bases. The same HMGA1a-binding sequence mutations introduced in the decoy oligoribonucleotides lost their competitive effect to restore exon inclusion (19). The authentic 5 splice site and the three potential pseudo 5 splice sites (a, b, and c) are indicated by arrows. (Bottom) The complexes on 32 P-labeled RNAs with HMGA1a and/or U1 snRNP analyzed by EMSA. Arrows indicate the discrete shifted complexes. Increasing amounts of purified U1 snRNP (0, 141, and 282 ng in 15 l) and recombinant HMGA1a (0, 191, and 383 ng in 15 l) are indicated by minus signs and triangles. Fixed amounts of U1 snRNP (282 ng in 15 l) and HMGA1a (287 ng in 15 l) are indicated by rectangles.
Source publication
Overexpression of high-mobility group A protein 1a (HMGA1a) causes aberrant exon 5 skipping of the Presenilin-2 (PS2) pre-mRNA,
which is almost exclusively detected in patients with sporadic Alzheimer's disease. An electrophoretic mobility shift assay
confirmed aberrant U1 small nuclear ribonucleoprotein particle (snRNP)-HMGA1a complex formation (v...
Contexts in source publication
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... for in vitro transcription. The template plasmid for BS1/5SS RNA (see Fig. 1) was constructed using an oligonucleotide-annealed DNA fragment of the following oligonucleotides: 5-agcttCTGGGCAAGTCTAGACGTAGTACC GCTGCTACAAGGTTGGTATCg-3 and 5-aattcGATACCAACCTTGTAGC AGCGGTACTACGTCTAGACTTGCCCAGa-3 (the HMGA1a-binding se- quence is underlined, and the parts of flanking HindIII and EcoRI sites are indicated in ...
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... pSP64 vector (Promega) to obtain pSP64-BS1/5SS. The plasmids for BS1/5SSmut and BS1mut/5SS RNAs (RNAs containing mu- tations at the 5 splice site and at the HMGA1a-binding sequence, respectively) were constructed in a similar way (pSP64-BS1/5SSmut and pSP64-BS1mut/5SS, respectively). The sequences of the transcribed RNAs are shown at the top of Fig. ...
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... To exclude the possibility of an additional factor(s) being neces- sary to form the HMGA1a-U1 snRNP complex, we used highly purified U1 snRNP and recombinant HMGA1a to perform an electrophoretic mobility shift assay (EMSA) with the short target RNA BS1/5SS, which contains a single copy of the HMGA1a binding sequence and a 5 splice site sequence (Fig. 1, top). We also prepared the variant RNAs BS1/5SSmut and BS1mut/5SS, which contain mutations at the 5 splice site and at the HMGA1a-binding sequence, respectively (Fig. 1, top). HMGA1a and U1 snRNP individually bound to the target BS1/5SS RNA in a dose-dependent manner and formed unique shifted complexes (Fig. 1, lanes 1 to 5). The addition ...
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... perform an electrophoretic mobility shift assay (EMSA) with the short target RNA BS1/5SS, which contains a single copy of the HMGA1a binding sequence and a 5 splice site sequence (Fig. 1, top). We also prepared the variant RNAs BS1/5SSmut and BS1mut/5SS, which contain mutations at the 5 splice site and at the HMGA1a-binding sequence, respectively (Fig. 1, top). HMGA1a and U1 snRNP individually bound to the target BS1/5SS RNA in a dose-dependent manner and formed unique shifted complexes (Fig. 1, lanes 1 to 5). The addition of both factors together led to the appearance of a super-shifted ternary complex (Fig. 1, lanes 6 and 7). The U1 snRNP and HMGA1a binding was not observed with BS1/5SSmut ...
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... and a 5 splice site sequence (Fig. 1, top). We also prepared the variant RNAs BS1/5SSmut and BS1mut/5SS, which contain mutations at the 5 splice site and at the HMGA1a-binding sequence, respectively (Fig. 1, top). HMGA1a and U1 snRNP individually bound to the target BS1/5SS RNA in a dose-dependent manner and formed unique shifted complexes (Fig. 1, lanes 1 to 5). The addition of both factors together led to the appearance of a super-shifted ternary complex (Fig. 1, lanes 6 and 7). The U1 snRNP and HMGA1a binding was not observed with BS1/5SSmut and BS1mut/5SS, respectively, and the super-shifted complex did not appear with either of these RNA fragments (Fig. 1, lanes 8 and 9). These results ...
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... mutations at the 5 splice site and at the HMGA1a-binding sequence, respectively (Fig. 1, top). HMGA1a and U1 snRNP individually bound to the target BS1/5SS RNA in a dose-dependent manner and formed unique shifted complexes (Fig. 1, lanes 1 to 5). The addition of both factors together led to the appearance of a super-shifted ternary complex (Fig. 1, lanes 6 and 7). The U1 snRNP and HMGA1a binding was not observed with BS1/5SSmut and BS1mut/5SS, respectively, and the super-shifted complex did not appear with either of these RNA fragments (Fig. 1, lanes 8 and 9). These results confirmed that the HMGA1a-U1 snRNP complex, in the absence of other factors, forms without steric hindrance on the minimal ...
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... unique shifted complexes (Fig. 1, lanes 1 to 5). The addition of both factors together led to the appearance of a super-shifted ternary complex (Fig. 1, lanes 6 and 7). The U1 snRNP and HMGA1a binding was not observed with BS1/5SSmut and BS1mut/5SS, respectively, and the super-shifted complex did not appear with either of these RNA fragments (Fig. 1, lanes 8 and 9). These results confirmed that the HMGA1a-U1 snRNP complex, in the absence of other factors, forms without steric hindrance on the minimal target RNA containing the essential elements, i.e., an HMGA1a-binding sequence and the adjacent 5 splice ...
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... site-like sequences in the original PS2 pre-mRNA (18) and also in the heterologous pre-mRNAs used previously (i.e., E1-BS1-E2 and E1-BS2-E2) in which HMGA1a-inducible exon skipping was reconstituted (19). On the other hand, our EMSA data indicated that the authentic 5 splice site is re- quired for the formation of the HMGA1a-U1 snRNP complex (Fig. ...
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... the above-described arguments, we sought to identify the actual U1 snRNA annealing site in the presence of excess HMGA1a using psoralen-mediated UV light cross-linking with the BS1/5SS RNA (derived from E1-BS1-E2), which con- tained three 5 splice site-like sequences (Fig. 1, top). We observed a single cross-linked product in a HeLa cell nuclear extract (Fig. 2A, lane 3), which was abolished either by tar- geted degradation of U1 snRNA (lane 5) or by mutation of the authentic 5 splice site (with BS1/5SSmut RNA) (lane 10), indicating specific cross-linking between U1 snRNA and the 5 splice site. Remarkably, ...
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... addition of recombinant HMGA1a did not diminish the U1 snRNA cross-linking signal, leading instead to a visible increase (Fig. 2A, lanes 6 and 7). We could not observe any other cross-linking signals with the BS1/5SS and BS1/5SSmut RNAs in the presence of HMGA1a due to expected U1 snRNP binding to the upstream 5 splice site-like sequences ( Fig. 2A, lanes 6, 7, 13, and 14). These results revealed that U1 snRNP binds to the authentic 5 splice site but not to the pseudo 5 splice sites, even in the presence of ...
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... binding, we examined the state of psoralen cross-linking using a time-course experiment. We found that HMGA1a induced a significant change in the ki- netics of U1 snRNA-5 splice site cross-linking with the BS1/ 5SS RNA substrate (Fig. 2B). The cross-linking was gradually diminished and almost disappeared at 60 min without the ad- dition of HMGA1a (Fig. 2B, lanes 1 to 3). In contrast, a substantial amount of the cross-linking was retained up to 60 min after the addition of HMGA1a (Fig. 2B, lanes 4 to 6). To examine whether the HMGA1a binding to the target site is a prerequisite for extended U1 snRNA-5 splice site cross-link- ing, we used the BS1mut/5SS RNA containing point muta- tions in the ...
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... contrast, a substantial amount of the cross-linking was retained up to 60 min after the addition of HMGA1a (Fig. 2B, lanes 4 to 6). To examine whether the HMGA1a binding to the target site is a prerequisite for extended U1 snRNA-5 splice site cross-link- ing, we used the BS1mut/5SS RNA containing point muta- tions in the HMGA1a-binding sequence (Fig. 1, top). With the addition of HMGA1a, the kinetics of cross-linking was similar 2222 OHE AND MAYEDA MOL. CELL. ...
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... HMGA1a-binding sequence in the PS2 pre-mRNA is lo- cated adjacent to the 5 splice site (Fig. 1, top) (18). Accordingly, we examined whether the distance between the HMGA1a-bind- ing site and the 5 splice site is critical to elicit the HMGA1 effect on extended U1 snRNA-5 splice site cross-linking. We prepared mmW/5SS, mWm/5SS, and Wmm/5SS RNAs, which contain three tandem sequences of either the wild-type HMGA1a binding sequence (W) or its ...
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... site cross-linking with the addition of HMGA1a (Fig. 4, lanes 3 to 8). Remarkably, the timely disso- FIG. 2. HMGA1a recruits U1 snRNP to the authentic 5 splice site and causes unusually extended binding. (A) Detection of psoralen- cross-linked U1 snRNA-5 splice site product (arrows) with 32 P-la- beled BS1/5SS RNA but not with BS1/5SSmut RNA (Fig. 1, top). Amounts of recombinant HMGA1a added (144 and 287 ng in 12.5 l) are indicated by triangles. U1 and U2 indicate digestion of endog- enous U1 and U2 snRNAs, respectively, using antisense oligonucle- otide-directed RNase H treatment. (B) Time course data from U1 snRNA psoralen cross-linking to the 5 splice site (arrow) using 32 P- labeled ...
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... HMGA1a added (144 and 287 ng in 12.5 l) are indicated by triangles. U1 and U2 indicate digestion of endog- enous U1 and U2 snRNAs, respectively, using antisense oligonucle- otide-directed RNase H treatment. (B) Time course data from U1 snRNA psoralen cross-linking to the 5 splice site (arrow) using 32 P- labeled BS1/5SS and BS1mut/5SS RNAs (Fig. 1, top). HMGA1a indicates the addition of recombinant HMGA1a (144 ng in 12.5 l) prior to ...
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... of the U1 snRNP from the 5 splice site was restored in the mWm/5SS and Wmm/5SS RNAs, even with the addition of HMGA1a (Fig. 4, lanes 9 to 14). This result demonstrates that the positioning of the original HMGA1a-binding site, which is adjacent to the downstream 5 splice site, is crucial for the functional contact between HMGA1a and the U1-70K component to promote extended U1 snRNP binding to the 5 splice site. These cross-linking experiments were performed using short RNA ...
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... that were not subjected to splicing. It was therefore important to examine whether the addition of HMGA1a also prevents U1 snRNP dissociation from the 5 splice site of a splicing-competent substrate during the splicing reaction. We used the BS1-E6 pre-mRNA, which led to the HMGA1a-induced inhibition of the constitutive splicing at the first step (Fig. 5A, lanes 1 to 6) (19). HMGA1a is not a general splicing inhibitor, as it had no significant effect on the splicing of the BS1mut-E6 pre-mRNA, which contains a mutated HMGA1a-binding sequence (Fig. 5A, lanes 7 to 12), or on the -globin pre-mRNA, which lacks an HMGA1a-binding se- quence ...
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... during the splicing reaction. We used the BS1-E6 pre-mRNA, which led to the HMGA1a-induced inhibition of the constitutive splicing at the first step (Fig. 5A, lanes 1 to 6) (19). HMGA1a is not a general splicing inhibitor, as it had no significant effect on the splicing of the BS1mut-E6 pre-mRNA, which contains a mutated HMGA1a-binding sequence (Fig. 5A, lanes 7 to 12), or on the -globin pre-mRNA, which lacks an HMGA1a-binding se- quence ...
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... time point indicated. The inhibition of splicing was monitored using denaturing PAGE, whereas the generated U1 snRNA-5 splice site cross-linked products were identified using a sensi- tive Northern blot analysis (Fig. 5B). We found that this cross- linking signal decreased gradually and almost disappeared at 90 min without the addition of HMGA1a (Fig. 5B, lanes 1 to 3), whereas the signal was retained up to 90 min after the addition of HMGA1a (Fig. 5B, lanes 4 to 6). These results led us to conclude that the aberrant HMGA1a-U1 snRNP com- plex prevents proper U1 snRNP dissociation from the 5 splice site during the active splicing reaction, leading to HMGA1a- binding site-dependent inhibition of ...
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... tether U1 snRNP. (Top) Aligned sequences of the target RNAs that contain the wild-type HMGA1a-binding sequence (W) and the mutated HMGA1a-binding sequence (m). The wild-type sequence is substituted with its mutated sequence to avoid potential effects of created cis-acting sequences. The first 14 nt are identical with those of BS1/5SS-series RNA (Fig. 1, top). (Bottom) Time course data obtained from U1 snRNA psoralen cross-linking to the 5 splice site (arrow) using these 32 P-labeled RNAs. HMGA1a indicates the addition of recombinant HMGA1a (191 ng in 12.5 l). U1 indicates digestion of endogenous U1 snRNA by antisense oligonucleotide-directed RNase H treatment (minus sign indicates the ...
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... antisense oligonucleotide-directed RNase H treatment (minus sign indicates the control lacking RNase treatment), which is also performed in the cross-linking with mWm and Wmm RNAs (data not shown). The asterisk indicates nonspecific by-products that were not cross-linked with U1 snRNA. a separation of the E complex from the nonspecific H complex (Fig. 6A, lanes 1 to 4), and the addition of HMGA1a markedly impaired the formation of the E complex (lanes 5 to 8). These results indicate that the HMGA1a-induced U1 snRNP reten- tion on the 5 splice site abrogated the ATP-independent for- mation of the E complex, blocking functional communication between 5 and 3 splice sites via SF1, the 35-kDa subunit of ...
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... (31); therefore, we analyzed the EDE complex using the iE5i RNA, which carries full exon 5 of PS2 with the flanking polypyrimidine tract, the 3 splice site, and the 5 splice sites (Fig. 6B, top). Native agarose gel analysis successfully detected a discrete complex separated from the nonspecific H complex that corresponded to the EDE complex (Fig. 6B, lanes 1 to 4). We observed the complete disruption of this specific EDE complex formation by the addition of HMGA1a (Fig. 6B, lanes 5 to 8). These results suggest that the HMGA1a-U1 snRNP complex arrests the exon definition com- plex across exon 5 in the PS2 pre-mRNA at an early ATP- independent stage, which is a crucial cause of HMGA1-in- duced ...
Citations
... Many proteins showed known connections to the mechanisms of AD pathology such as amyloid or Tau pathways including secretases and presenilin. For example HMGA1 induces aberrant exon skipping of Presenilin-2 (PS2) RNA, in sporadic Alzheimer's disease [93]. A chymotrypsin like activity results in a carboxyl-terminal-truncated Apolipoprotein E4 that causes Alzheimer's disease-Like Neurodegeneration in mice [94]. ...
Background
A practical strategy to discover proteins specific to Alzheimer’s dementia (AD) may be to compare the plasma peptides and proteins from patients with dementia to normal controls and patients with neurological conditions like multiple sclerosis or other diseases. The aim was a proof of principle for a method to discover proteins and/or peptides of plasma that show greater observation frequency and/or precursor intensity in AD. The endogenous tryptic peptides of Alzheimer’s were compared to normals, multiple sclerosis, ovarian cancer, breast cancer, female normal, sepsis, ICU Control, heart attack, along with their institution-matched controls, and normal samples collected directly onto ice.
Methods
Endogenous tryptic peptides were extracted from blinded, individual AD and control EDTA plasma samples in a step gradient of acetonitrile for random and independent sampling by LC–ESI–MS/MS with a set of robust and sensitive linear quadrupole ion traps. The MS/MS spectra were fit to fully tryptic peptides within proteins identified using the X!TANDEM algorithm. Observation frequency of the identified proteins was counted using SEQUEST algorithm. The proteins with apparently increased observation frequency in AD versus AD Control were revealed graphically and subsequently tested by Chi Square analysis. The proteins specific to AD plasma by Chi Square with FDR correction were analyzed by the STRING algorithm. The average protein or peptide log 10 precursor intensity was compared across disease and control treatments by ANOVA in the R statistical system.
Results
Peptides and/or phosphopeptides of common plasma proteins such as complement C2, C7, and C1QBP among others showed increased observation frequency by Chi Square and/or precursor intensity in AD. Cellular gene symbols with large Chi Square values (χ2 ≥ 25, p ≤ 0.001) from tryptic peptides included KIF12, DISC1, OR8B12, ZC3H12A, TNF, TBC1D8B, GALNT3, EME2, CD1B, BAG1, CPSF2, MMP15, DNAJC2, PHACTR4, OR8B3, GCK, EXOSC7, HMGA1 and NT5C3A among others. Similarly, increased frequency of tryptic phosphopeptides were observed from MOK, SMIM19, NXNL1, SLC24A2, Nbla10317, AHRR, C10orf90, MAEA, SRSF8, TBATA, TNIK, UBE2G1, PDE4C, PCGF2, KIR3DP1, TJP2, CPNE8, and NGF amongst others. STRING analysis showed an increase in cytoplasmic proteins and proteins associated with alternate splicing, exocytosis of luminal proteins, and proteins involved in the regulation of the cell cycle, mitochondrial functions or metabolism and apoptosis. Increases in mean precursor intensity of peptides from common plasma proteins such as DISC1, EXOSC5, UBE2G1, SMIM19, NXNL1, PANO, EIF4G1, KIR3DP1, MED25, MGRN1, OR8B3, MGC24039, POLR1A, SYTL4, RNF111, IREB2, ANKMY2, SGKL, SLC25A5, CHMP3 among others were associated with AD. Tryptic peptides from the highly conserved C-terminus of DISC1 within the sequence MPGGGPQGAPAAAGGGGVSHRAGSRDCLPPAACFR and ARQCGLDSR showed a higher frequency and highest intensity in AD compared to all other disease and controls.
Conclusion
Proteins apparently expressed in the brain that were directly related to Alzheimer’s including Nerve Growth Factor (NFG), Sphingomyelin Phosphodiesterase, Disrupted in Schizophrenia 1 (DISC1), the cell death regulator retinitis pigmentosa (NXNl1) that governs the loss of nerve cells in the retina and the cell death regulator ZC3H12A showed much higher observation frequency in AD plasma vs the matched control. There was a striking agreement between the proteins known to be mutated or dis-regulated in the brains of AD patients with the proteins observed in the plasma of AD patients from endogenous peptides including NBN, BAG1, NOX1, PDCD5, SGK3, UBE2G1, SMPD3 neuronal proteins associated with synapse function such as KSYTL4, VTI1B and brain specific proteins such as TBATA.
... Pathological evidence of human brain-insoluble proteome has demonstrated the unique aggregation pattern of U1 snRNPs in AD diseased neuronal cells 23 , causing global splicing defects in the early stages of AD in the affected patients. The splicing pattern of its downstream target, Presenilin-2 (PS2) is affected by an exon skipping event at exon 5, an event constantly detected in AD patients 24 . Intriguingly, aggregated proteolytic products of U1-70K have been implicated in neuronal toxicity in AD as well. ...
Eukaryotic cells can expand their coding ability by using their splicing machinery, spliceosome, to process precursor mRNA (pre-mRNA) into mature messenger RNA. The mega-macromolecular spliceosome contains multiple subcomplexes, referred to as small nuclear ribonucleoproteins (snRNPs). Among these, U1 snRNP and its central component, U1-70K, are crucial for splice site recognition during early spliceosome assembly. The human U1-70K has been linked to several types of human autoimmune and neurodegenerative diseases. However, its phylogenetic relationship has been seldom reported. To this end, we carried out a systemic analysis of 95 animal U1-70K genes and compare these proteins to their yeast and plant counterparts. Analysis of their gene and protein structures, expression patterns and splicing conservation suggest that animal U1-70Ks are conserved in their molecular function, and may play essential role in cancers and juvenile development. In particular, animal U1-70Ks display unique characteristics of single copy number and a splicing isoform with truncated C-terminal, suggesting the specific role of these U1-70Ks in animal kingdom. In summary, our results provide phylogenetic overview of U1-70K gene family in vertebrates. In silico analyses conducted in this work will act as a reference for future functional studies of this crucial U1 splicing factor in animal kingdom.
... Many proteins showed known connections to the mechanisms of AD pathology including roles in the Amyloid or Tau pathways including secretases and presenilin. For example HMGA1 induces aberrant exon skipping of Presenilin-2 (PS2) RNA, in sporadic Alzheimer's disease [88]. A chymotrypsin like activity results in a carboxyl-terminal-truncated Apolipoprotein E4 that causes Alzheimer's disease-Like Neurodegeneration in mice [89]. ...
BackgroundA practical strategy to discover proteins specific to Alzheimer’s dementia (AD) may be to compare the plasma peptides and proteins from patients with dementia to normal controls and patients with neurological conditions like multiple sclerosis or other diseases. The aim was a proof of principle for a method to discover proteins and/or peptides of plasma that show greater observation frequency and/or precursor intensity in AD. The endogenous tryptic peptides of Alzheimer’s were compared to normals, multiple sclerosis, ovarian cancer, breast cancer, female normal, sepsis, ICU Control, heart attack, along with their institution-matched controls, and normal samples collected directly onto ice.Methods
Endogenous tryptic peptides were extracted from blinded, individual AD and control EDTA plasma samples in a step gradient of acetonitrile for random and independent sampling by LC-ESI-MS/MS with a set of robust and sensitive linear quadrupole ion traps. The MS/MS spectra were fit to fully tryptic peptides within proteins identified using the X!TANDEM algorithm. Observation frequency of the identified proteins was counted using SEQUEST algorithm. The proteins with apparently increased observation frequency in AD versus AD Control were revealed graphically and subsequently tested by Chi Square analysis. The proteins specific to AD plasma by Chi Square with FDR correction were analyzed by the STRING algorithm. The average protein or peptide log 10 precursor intensity was compared across disease and control treatments by ANOVA in the R statistical system. ResultsPeptides and/or phosphopeptides of common plasma proteins such as complement C2, C7, and C1QBP among others showed increased observation frequency by Chi Square and/or precursor intensity in AD. Cellular gene symbols with large Chi Square values (χ2 ≥ 25, p ≤ 0.001) from tryptic peptides included KIF12, DISC1, OR8B12, ZC3H12A, TNF, TBC1D8B, GALNT3, EME2, CD1B, BAG1, CPSF2, MMP15, DNAJC2, PHACTR4, OR8B3, GCK, EXOSC7, HMGA1 and NT5C3A among others. Similarly, increased frequency of tryptic phosphopeptides were observed from MOK, SMIM19, NXNL1, SLC24A2, Nbla10317, AHRR, C10orf90, MAEA, SRSF8, TBATA, TNIK, UBE2G1, PDE4C, PCGF2, KIR3DP1, TJP2, CPNE8, and NGF amongst others. STRING analysis showed an increase in cytoplasmic proteins and proteins associated with alternate splicing, exocytosis of luminal proteins, and proteins involved in the regulation of the cell cycle, mitochondrial functions or metabolism and apoptosis. Increases in mean precursor intensity of peptides from common plasma proteins such as DISC1, EXOSC5, UBE2G1, SMIM19, NXNL1, PANO, EIF4G1, KIR3DP1, MED25, MGRN1, OR8B3, MGC24039, POLR1A, SYTL4, RNF111, IREB2, ANKMY2, SGKL, SLC25A5, CHMP3 among others were associated with AD. Tryptic peptides from the highly conserved C-terminus of DISC1 within the sequence MPGGGPQGAPAAAGGGGVSHRAGSRDCLPPAACFR and ARQCGLDSR showed a higher frequency and highest intensity in AD compared to all other disease and controls. Conclusion
Proteins apparently from the brain that were directly related to Alzheimer’s including Nerve Growth Factor (NFG), Sphingomyelin Phosphodiesterase, Disrupted in Schizophrenia 1 (DISC1), the cell death regulator retinitis pigmentosa (NXNl1) that governs the loss of nerve cells in the retina and the cell death regulator ZC3H12A showed much higher observation frequency in AD plasma. There was a striking agreement between the proteins known to be mutated or dis-regulated in the brains of AD patients with the proteins observed in the plasma of AD patients from endogenous peptides including NBN, BAG1, NOX1, PDCD5, SGK3, UBE2G1, SMPD3 neuronal proteins associated with synapse function such as KSYTL4, VTI1B and brain specific proteins such as TBATA.
... In vitro studies have shown that HMGA1 binds to a site within exon 5 and inactivates normal splicing, leading to the generation of a truncated presenilin 2 protein [33]. The increased levels of HMGA1 proteins in patients with sporadic AD is likely due to hypoxia in neuronal cells [30][31][32]34,35]. Hypoxia would seem to favour the accumulation of amyloid β, as well as impairing tau phosphorylation and contributing to the degeneration of neurons and promoting the innate immune system [36], thus perpetuating the vicious circle that promotes the pathogenesis of AD. ...
The intricate relationships between innate immunity and brain diseases raise increased interest across the wide spectrum of neurodegenerative and neuropsychiatric disorders. Barriers, such as the blood–brain barrier, and innate immunity cells such as microglia, astrocytes, macrophages, and mast cells are involved in triggering disease events in these groups, through the action of many different cytokines. Chronic inflammation can lead to dysfunctions in large-scale brain networks. Neurodegenerative diseases, such as Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, amyotrophic lateral sclerosis, and frontotemporal dementia, are associated with a substrate of dysregulated immune responses that impair the central nervous system balance. Recent evidence suggests that similar phenomena are involved in psychiatric diseases, such as depression, schizophrenia, autism spectrum disorders, and post-traumatic stress disorder. The present review summarizes and discusses the main evidence linking the innate immunological response in neurodegenerative and psychiatric diseases, thus providing insights into how the responses of innate immunity represent a common denominator between diseases belonging to the neurological and psychiatric sphere. Improved knowledge of such immunological aspects could provide the framework for the future development of new diagnostic and therapeutic approaches.
... In particular, U1-70K functions as a central unit in this snRNP. Increasing evidence has demonstrated that alterations in U1-70K expression results in various human diseases, including Alzheimer's disease (Ohe and Mayeda, 2010;Bai et al., 2013), systemic lupus erythematosus (Yao et al., 1994;Monneaux et al., 2007), cytomegalovirus infection (Newkirk et al., 2001), mixed connective tissue disease (Hof et al., 2005) and amyotrophic lateral sclerosis (Yu et al., 2015), suggesting the importance of this protein in maintaining normal cellular function. In plants, however, many fewer reports have been published to elucidate its biological function. ...
Intron‐containing genes have the ability to generate multiple transcript isoforms by splicing, thereby greatly expanding eukaryotic transcriptome and proteome. In eukaryotic cells, precursor mRNA (pre‐mRNA) splicing is performed by a mega‐macromolecular complex defined as a spliceosome. Among its splicing components, U1 small nuclear ribonucleoprotein (U1 snRNP) is the smallest subcomplex involved in early spliceosome assembly and 5’‐splice site recognition. Its central component, named as U1‐70K, has been extensively characterized in animals and yeast. However, very few investigations on U1‐70K genes have been conducted in plants. To this end, we performed a comprehensive study to systematically identify 115 U1‐70K genes from 67 plant species, ranging from algae to angiosperms. Phylogenetic analysis suggested that expansion of plant U1‐70K gene family was likely driven by whole genome duplications. Subsequent comparisons of gene structures, protein domains, promoter regions and conserved splicing patterns indicated that plant U1‐70Ks are likely to preserve their conserved molecular function across plant lineage, and play an important functional role in response to environmental stresses. Furthermore, genetic analysis by using T‐DNA insertion mutants suggested that Arabidopsis U1‐70K may be involved in response to osmotic stress. Our results provide a general overview of this gene family in Viridiplantae and will act as a reference source to carry out future mechanistic studies on this U1 snRNP‐specific splicing factor.
... The accumulation of U1 snRNP resulted in changes in RNA splicing [89,90]. Aberrant splicing in presenilin 2 is present in sporadic ADD patients [91]. Furthermore, altered splicing of the receptor for advanced glycation end products (RAGE) occurs in ADD [92]. ...
Background:
Dementia is a major public health concern affecting approximately 47 million people worldwide. Mild cognitive impairment (MCI) is one form of dementia that affects an individual's memory with or without affecting their daily life. Alzheimer's disease dementia (ADD) is a more severe form of dementia that usually affects elderly individuals. It remains unclear whether MCI is a distinct disorder from or an early stage of ADD.
Methods:
Gene expression data from blood were analyzed to identify potential biomarkers that may be useful for distinguishing between these two forms of dementia.
Results:
A meta-analysis revealed 91 genes dysregulated in individuals with MCI and 387 genes dysregulated in ADD. Pathway analysis identified seven pathways shared between MCI and ADD and nine ADD-specific pathways. Fifteen transcription factors were associated with MCI and ADD, whereas seven transcription factors were specific for ADD. Mir-335-5p was specific for ADD, suggesting that it may be useful as a biomarker. Diseases that are associated with MCI and ADD included developmental delays, cognition impairment, and movement disorders.
Conclusion:
These results provide a better molecular understanding of peripheral changes that occur in MCI and ADD patients and may be useful in the identification of diagnostic and prognostic biomarkers.
... In PS2, deletion of exon 5 expresses truncated PS2 isoform, PS2V which accumulates rapidly in the hippocampus of patients with sporadic AD. This is due to the overexpression of high-mobility group A protein 1a (HMGA1a), a sequence-specific RNA-binding factor [107]. Another protein of interest in AD is GFAP (glial fibrillary acidic protein), its variant, GFAP + 1 was found in AD brains due to the expression of two novel splice forms: lacking exon 6 or deletion of 164 nt [108]. ...
Neurodegenerative diseases are usually sporadic in nature and commonly influenced by a wide range of genetic, life style and environmental factors. A unifying feature of Alzheimer’s disease (AD) and Parkinson’s disease (PD) is the abnormal accumulation and processing of mutant or damaged intra and extracellular proteins; this leads to neuronal vulnerability and dysfunction in the brain. Through a detailed review of ubiquitin proteasome, mRNA splicing, mitochondrial dysfunction, and oxidative stress pathway interrelation on neurodegeneration can improve the understanding of the disease mechanism. The identified pathways common to AD and PD nominate promising new targets for further studies, and as well as biomarkers. These insights suggested would likely provide major stimuli for developing unified treatment approaches to combat neurodegeneration. More broadly, pathways can serve as vehicles for integrating findings from diverse studies of neurodegeneration. The evidence examined in this review provides a brief overview of the current literature on significant pathways in promoting in AD, PD. Additionally, these insights suggest that biomarkers and treatment strategies may require simultaneous targeting of multiple components.
... The 5' splice site of the hGRα variant exon 1-D has a HMGA1a RNA-binding site adjacently upstream the 5' splice site (5'-UGCCGCACAAGgu-3'). This hGRα HMGA1a RNA-binding site resembles the original HMGA1a RNAbinding site adjacently upstream the 5' splice site of hPS2 exon 5 (5'-GCUGCUACAAGgu-3') [9,10] and the one adjacently upstream the pseudo-5' splice site of hERα exon 1 (5'-GCGGCUACACGgu-3') [7,8] with a GC-rich stretch followed by common nucleotides shown in bold and underlined ( Figure 2A). It has recently been reported that HMGA1a binds stems of 7SK RNA (5'---CCG---loop---CGG---3'; the HMGA1a binding site is underlined) [11] ( Figure 2B). ...
... Indeed, it was the trapping of U1 snRNP by HMA1a that made HMGA1a inhibit splicing and induce exon-skipping. Remarkably, this effect was only found when the HMGA1a RNA binding site was adjacent the 5' splice site [10]. In a search for other targets for HMGA1a, the human estrogen receptor alpha (hERα) gene came to our minds, because a vast list of literature had been reporting the relation of hERα and HMGA1a in breast cancer cells. ...
... We transiently expressed a decoy of the HMGA1a RNA binding site (bold) with a microRNA nucleus localization signal (underlined) at its 3' end (5'-GCUGCUACAAGAGUGUU [16]. This HMGA1a RNA decoy efficiently blocked HMGA1a-induced skipping of hERα exon1 [10]. We also pursued the mechanism of this exon-skipping. ...
... dissociation of U1 snRNP from the spliceosome and induces aberrant splicing of PS2 exon 5 (Ohe and Mayeda, 2010). This effect was also found in HIV-1 splice site regulation (Tsuruno et al., 2011). ...
... EMSA was performed as previously described (Ohe and Mayeda, 2010) with the following minor modifications. Each reaction mixture was incubated at 30 • C for 15 min and RNAprotein complexes were analyzed by 5% polyacrylamide gel electrophoresis (PAGE) (acrylamide: bisacrylamide ratio 30:1 [wt/wt]) at 4 • C. ...
... In vitro splicing was performed as described (Ohe and Mayeda, 2010), with minor modifications. 32 P-labeled pre-mRNA (∼20 fmol) was incubated at 30 • C for 2 h in a 12.5 µl reaction mixture containing 3 mM ATP, 20 mM creatine phosphate, 20 mM HEPES-NaOH (pH 7.3), 3.5 mM MgCl 2 , 2% (wt/vol) low-molecular-weight polyvinyl alcohol (Sigma), and 3.5 µl of HeLa cell nuclear extract (CilBiotech). ...
Objectives: The high-mobility group A protein 1a (HMGA1a) protein is known as a transcription factor that binds to DNA, but recent studies have shown it exerts novel functions through RNA-binding. We were prompted to decipher the mechanism of HMGA1a-induced alternative splicing of the estrogen receptor alpha (ERα) that we recently reported would alter tamoxifen sensitivity in MCF-7 TAMR1 cells.
Methods: Endogenous expression of full length ERα66 and its isoform ERα46 were evaluated in MCF-7 breast cancer cells by transient expression of HMGA1a and an RNA decoy (2′-O-methylated RNA of the HMGA1a RNA-binding site) that binds to HMGA1a. RNA-binding of HMGA1a was checked by RNA-EMSA. In vitro splicing assay was performed to check the direct involvement of HMGA1a in splicing regulation. RNA-EMSA assay in the presence of purified U1 snRNP was performed with psoralen UV crosslinking to check complex formation of HMGA1a-U1 snRNP at the upstream pseudo-5′ splice site of exon 1.
Results: HMGA1a induced exon skipping of a shortened exon 1 of ERα in in vitro splicing assays that was blocked by the HMGA1a RNA decoy and sequence-specific RNA-binding was confirmed by RNA-EMSA. RNA-EMSA combined with psoralen UV crosslinking showed that HMGA1a trapped purified U1 snRNP at the upstream pseudo-5′ splice site.
Conclusions: Regulation of ERα alternative splicing by an HMGA1a-trapped U1 snRNP complex at the upstream 5′ splice site of exon 1 offers novel insight on 5′ splice site regulation by U1 snRNP as well as a promising target in breast cancer therapy where alternative splicing of ERα is involved.
... Thus, we searched for an HMGA1a RNAbinding sequence [13] (5′-GCUGCUACAAG-3′) in the ERα gene. Since HMGA1a functions only when it is adjacent a 5′ splice site sequence [15], the search was performed by attaching a GU dinucleotide to the 3′ end of the HMGA1a RNA-binding sequence. Using a web-based program, Clustal Omega (http://www.ebi.ac.uk/Tools/msa/clustalo/), we aligned genomic sequence of the ERα gene and 5′-GCUGCUACAAGGU-3′ (note that the bold underlined GU dinucleotide is attached). ...
... During extensive analyses of the RNA-binding function of HMGA1a [13,15,25], we found an RNA element in the ERα gene where HMGA1a may function by regulating alternative splicing. The HMGA1a-binding sequence needs to be adjacently upstream a 5′ splice site to trap and inhibit normal dissociation of U1 snRNP during spliceosome assembly [15]. ...
... During extensive analyses of the RNA-binding function of HMGA1a [13,15,25], we found an RNA element in the ERα gene where HMGA1a may function by regulating alternative splicing. The HMGA1a-binding sequence needs to be adjacently upstream a 5′ splice site to trap and inhibit normal dissociation of U1 snRNP during spliceosome assembly [15]. We found an HMGA1a-binding sequence that fulfills these requirements in ERα exon 1 located 33 nucleotides upstream the authentic-5′ splice site and also adjacently upstream a pseudo-5′ splice site (Fig.1A). ...
The high-mobility group A protein 1a (HMGA1a) protein is known as an oncogene whose expression level in cancer tissue correlates with the malignant potential, and known as a component of senescence-related structures connecting it to tumor suppressor networks in fibroblasts. HMGA1 protein binds to DNA, but recent studies have shown it exerts novel functions through RNA-binding. Our previous studies have shown that sequence-specific RNA-binding of HMGA1a induces exon-skipping of Presenilin-2 exon 5 in sporadic Alzheimer disease. Here we show that HMGA1a induced exon-skipping of the estrogen receptor alpha (ERα) gene and increased ERα46 mRNA expression in MCF-7 breast cancer cells. An RNA-decoy of HMGA1a efficiently blocked this event and reduced ERα46 protein expression. Blockage of HMGA1a RNA-binding property consequently induced cell growth through reduced ERα46 expression in MCF-7 cells and increased sensitivity to tamoxifen in the tamoxifen-resistant cell line, MCF-7/TAMR1. Stable expression of an HMGA1a RNA-decoy in MCF-7 cells exhibited decreased ERα46 protein expression and increased estrogen-dependent tumor growth when these cells were implanted in nude mice. These results show HMGA1a is involved in alternative splicing of the ERα gene and related to estrogen-related growth as well as tamoxifen sensitivity in MCF-7 breast cancer cells.
