scholarly journals Activation of Cryptic 3′ Splice-Sites by SRSF2 Contributes to Cassette Exon Skipping

Cells ◽  
2019 ◽  
Vol 8 (7) ◽  
pp. 696 ◽  
Author(s):  
Heegyum Moon ◽  
Ha Na Jang ◽  
Yongchao Liu ◽  
Namjeong Choi ◽  
Jagyeong Oh ◽  
...  
Keyword(s):  
Splice Site ◽  
Binding Sites ◽  
Exon Skipping ◽  
Cassette Exon ◽  
Intron Splicing ◽  
Splice Sites ◽  
Polypyrimidine Tract ◽  
Associated Sequences ◽  
Cryptic Splice Sites ◽  
Survival Of Motor Neuron

Here we show that the serine/arginine rich splicing factor 2 (SRSF2) promotes cryptic 3′ splice-site (3′AG′) usage during cassette exon exclusion in survival of motor neuron (SMN2) minigenes. Deletion of the 3′AG′ (3′AG′1), its associated branch point (BP′) and polypyrimidine tract (PPT′) sequences directs SRSF2 to promote a second 3′AG′ (3′AG′2) with less conserved associated region for intron splicing. Furthermore, deletion of both 3′AG′1 and 3′AG′2 and their associated sequences triggered usage of a third 3′AG′3 that has very weak associated sequences. Interestingly, when intron splicing was directed to the 3′AG′ cryptic splice-sites, intron splicing from the canonical 3′AG splice-site was reduced along with a decrease in cassette exon inclusion. Moreover, multiple SRSF2 binding sites within the intron are responsible for 3′AG′ activation. We conclude that SRSF2 facilitates exon exclusion by activating a cryptic 3′AG′ and inhibiting downstream intron splicing.

Characterisation and quantification of F8 transcripts of ten putative splice site mutations

2015 ◽  
Vol 113 (03) ◽  
pp. 585-592 ◽  
Author(s):  
Yeling Lu ◽  
Yufeng Ruan ◽  
Qiulan Ding ◽  
Xuefeng Wang ◽  
Xiaodong Xi ◽  
...  
Keyword(s):  
Splice Site ◽  
Mrna Level ◽  
Gene Mutations ◽  
Exon Skipping ◽  
Splice Sites ◽  
Wild Type ◽  
Rt Pcr ◽  
Reverse Transcription Pcr ◽  
Fluorescent Pcr ◽  
Cryptic Splice Sites

SummaryMutations affecting splice sites comprise approximately 7.5 % of the known F8 gene mutations but only a few were verified at mRNA level. In the present study, 10 putative splice site mutations were characterised by mRNA analysis using reverse transcription PCR (RT-PCR). Quantitative real-time RT-PCR (RT-qPCR) and co-amplification fluorescent PCR were used in combination to quantify the amount of each of multiple F8 transcripts. All of the mutations resulted in aberrant splicing. One of them (c.6187+1del1) generated one form of F8 transcript with exon skipping, and the remaining nine mutations (c.602-6T>C, c.1752+5_1752+6insGTTAG, c.1903+5G>A, c.5219+3A>G, c.5586+3A>T, c.969A>T, c.265+4A>G, c.601+1_601+5del5 and c.1444-8_1444del9) produced multiple F8 transcripts with exon skipping, activation of cryptic splice site and/or normal splicing. Residual wild-type F8 transcripts were produced by the first six of the nine mutations with amounts of 3.9 %>, 14.2 %>, 5.2 %>, 19.2 %>, 1.8 °% and 2.5 %> of normal levels, respectively, which were basically consistent with coagulation phenotypes in the related patients. In comparison with the mRNA findings, software Alamut v2.3 had values in the prediction of pathogenic effects on native splice sites but was not reliable in the prediction of activation of cryptic splice sites. Our quantification of F8 transcripts may provide an alternative way to evaluate the low expression levels of residue wild-type F8 transcripts and help to explain the severity of haemophilia A caused by splicing site mutations.

Control of adenovirus E1B mRNA synthesis by a shift in the activities of RNA splice sites

1984 ◽  
Vol 4 (5) ◽  
pp. 966-972
Author(s):  
C Montell ◽  
E F Fisher ◽  
M H Caruthers ◽  
A J Berk
Keyword(s):  
Splice Site ◽  
Rna Splicing ◽  
Early Phase ◽  
Late Phase ◽  
Productive Infection ◽  
Splice Sites ◽  
Mrna Synthesis ◽  
Adenovirus E1b ◽  
Cryptic Splice Sites ◽  
Rna Splice Sites

The primary transcript from adenovirus 2 early region 1B (E1B) is processed by differential RNA splicing into two overlapping mRNAs, 13S and 22S. The 22S mRNA is the major E1B mRNA during the early phase of infection, whereas the 13S mRNA predominates during the late phase. In previous work, it has been shown that this shift in proportions of the E1B mRNAs is influenced by increased cytoplasmic stability of the 13S mRNA at late times in infection. Two observations presented here demonstrate that the increase in proportion of the 13S mRNA at late times is also regulated by a change in the specificity of RNA splicing. First, the relative concentrations of the 13S to 22S nuclear RNAs were not constant throughout infection but increased at late times. Secondly, studies with the mutant, adenovirus 2 pm2250 , provided evidence that there was an increased propensity to utilize a 5' splice in the region of the 13S 5' splice site at late times in infection. Adenovirus 2 pm2250 has a G----C transversion in the first base of E1B 13S mRNA intron preventing splicing of the 13S mRNA but not of the 22S mRNA. During the early phase of a pm2250 infection, the E1B primary transcripts were processed into the 22S mRNA only. However, during the late phase, when the 13S mRNA normally predominates, E1B primary transcripts were also processed by RNA splicing at two formerly unused or cryptic 5' splice sites. Both cryptic splice sites were located much closer to the disrupted 13S 5' splice site than to the 22S 5' splice site. Thus, the temporal increase in proportion of the 13S mRNA to the 22S mRNA is regulated by two processes, an increase in cytoplasmic stability of the 13S mRNA and an increased propensity to utilize the 13S 5' splice site during the late phase of infection. Adenovirus 2 pm2250 was not defective for productive infection of HeLa cells or for transformation of rat cells.

scholarly journals In vivo recognition of a vertebrate mini-exon as an exon-intron-exon unit.

1993 ◽  
Vol 13 (5) ◽  
pp. 2677-2687 ◽  
Author(s):  
D A Sterner ◽  
S M Berget
Keyword(s):  
Splice Site ◽  
Rna Splicing ◽  
Troponin I ◽  
Exon Skipping ◽  
Splice Sites ◽  
Small Vertebrate ◽  
Upstream Exon ◽  
I Gene

Very small vertebrate exons are problematic for RNA splicing because of the proximity of their 3' and 5' splice sites. In this study, we investigated the recognition of a constitutive 7-nucleotide mini-exon from the troponin I gene that resides quite close to the adjacent upstream exon. The mini-exon failed to be included in spliced RNA when placed in a heterologous gene unless accompanied by the upstream exon. The requirement for the upstream exon disappeared when the mini-exon was internally expanded, suggesting that the splice sites bordering the mini-exon are compatible with those of other constitutive vertebrate exons and that the small size of the exon impaired inclusion. Mutation of the 5' splice site of the natural upstream exon did not result in either exon skipping or activation of a cryptic 5' splice site, the normal vertebrate phenotypes for such mutants. Instead, a spliced RNA accumulated that still contained the upstream intron. In vitro, the mini-exon failed to assemble into spliceosome complexes unless either internally expanded or accompanied by the upstream exon. Thus, impaired usage of the mini-exon in vivo was accompanied by impaired recognition in vitro, and recognition of the mini-exon was facilitated by the presence of the upstream exon in vivo and in vitro. Cumulatively, the atypical in vivo and in vitro properties of the troponin exons suggest a mechanism for the recognition of this mini-exon in which initial recognition of an exon-intron-exon unit is followed by subsequent recognition of the intron.

scholarly journals Multiple Splicing Defects in an Intronic False Exon

2000 ◽  
Vol 20 (17) ◽  
pp. 6414-6425 ◽  
Author(s):  
Hanzhen Sun ◽  
Lawrence A. Chasin
Keyword(s):  
Splice Site ◽  
Negative Influence ◽  
Splice Sites ◽  
Polypyrimidine Tract ◽  
Exon Definition ◽  
Exon 2 ◽  
Vast Number ◽  
Consensus Sequences ◽  
Splice Site Sequence ◽  
Necessary And Sufficient

ABSTRACT Splice site consensus sequences alone are insufficient to dictate the recognition of real constitutive splice sites within the typically large transcripts of higher eukaryotes, and large numbers of pseudoexons flanked by pseudosplice sites with good matches to the consensus sequences can be easily designated. In an attempt to identify elements that prevent pseudoexon splicing, we have systematically altered known splicing signals, as well as immediately adjacent flanking sequences, of an arbitrarily chosen pseudoexon from intron 1 of the human hprt gene. The substitution of a 5′ splice site that perfectly matches the 5′ consensus combined with mutation to match the CAG/G sequence of the 3′ consensus failed to get this model pseudoexon included as the central exon in a dhfr minigene context. Provision of a real 3′ splice site and a consensus 5′ splice site and removal of an upstream inhibitory sequence were necessary and sufficient to confer splicing on the pseudoexon. This activated context also supported the splicing of a second pseudoexon sequence containing no apparent enhancer. Thus, both the 5′ splice site sequence and the polypyrimidine tract of the pseudoexon are defective despite their good agreement with the consensus. On the other hand, the pseudoexon body did not exert a negative influence on splicing. The introduction into the pseudoexon of a sequence selected for binding to ASF/SF2 or its replacement with β-globin exon 2 only partially reversed the effect of the upstream negative element and the defective polypyrimidine tract. These results support the idea that exon-bridging enhancers are not a prerequisite for constitutive exon definition and suggest that intrinsically defective splice sites and negative elements play important roles in distinguishing the real splicing signal from the vast number of false splicing signals.

scholarly journals Acute depletion of METTL3 identifies a role for N6-methyladenosine in alternative intron/exon inclusion in the nascent transcriptome

2020 ◽  
Author(s):  
Guifeng Wei ◽  
Mafalda Almeida ◽  
Greta Pintacuda ◽  
Heather Coker ◽  
Joseph S Bowness ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Histone Modification ◽  
Splice Site ◽  
Rna Processing ◽  
Binding Sites ◽  
Rna Binding ◽  
Embryonic Stem ◽  
Splice Sites ◽  
Rna Regulation

AbstractRNA N6-methyladenosine (m6A) modification plays important roles in multiple aspects of RNA regulation. m6A is installed co-transcriptionally by the METTL3/14 complex, but its direct roles in RNA processing remain unclear. Here we investigate the presence of m6A in nascent RNA of mouse embryonic stem cells (mESCs). We find that around 10% m6A peaks are in introns, often close to 5’-splice sites. RNA m6A peaks significantly overlap with RBM15 RNA binding sites and the histone modification H3K36me3. Interestingly, acute dTAG depletion of METTL3 reveals that inclusion of m6A-bearing alternative introns/exons in the nascent transcriptome is disrupted. For terminal or variable-length exons, m6A peaks are generally located upstream of a repressed 5’-splice site, and downstream of an enhanced 5’-splice site. Intriguingly, genes with the most immediate effects on splicing include several components of the m6A pathway, suggesting an autoregulatory function. Our findings demonstrate a direct crosstalk between m6A machinery and the regulation of RNA processing.

Exon definition may facilitate splice site selection in RNAs with multiple exons

1990 ◽  
Vol 10 (1) ◽  
pp. 84-94 ◽  
Author(s):  
B L Robberson ◽  
G J Cote ◽  
S M Berget
Keyword(s):  
Splice Site ◽  
Site Selection ◽  
Exon Skipping ◽  
Splice Sites ◽  
Splice Site Selection ◽  
Spliceosome Assembly ◽  
Exon Definition ◽  
Exon 2 ◽  
Correct Orientation

Interactions at the 3' end of the intron initiate spliceosome assembly and splice site selection in vertebrate pre-mRNAs. Multiple factors, including U1 small nuclear ribonucleoproteins (snRNPs), are involved in initial recognition at the 3' end of the intron. Experiments were designed to test the possibility that U1 snRNP interaction at the 3' end of the intron during early assembly functions to recognize and define the downstream exon and its resident 5' splice site. Splicing precursor RNAs constructed to have elongated second exons lacking 5' splice sites were deficient in spliceosome assembly and splicing activity in vitro. Similar substrates including a 5' splice site at the end of exon 2 assembled and spliced normally as long as the second exon was less than 300 nucleotides long. U2 snRNPs were required for protection of the 5' splice site terminating exon 2, suggesting direct communication during early assembly between factors binding the 3' and 5' splice sites bordering an exon. We suggest that exons are recognized and defined as units during early assembly by binding of factors to the 3' end of the intron, followed by a search for a downstream 5' splice site. In this view, only the presence of both a 3' and a 5' splice site in the correct orientation and within 300 nucleotides of one another will stable exon complexes be formed. Concerted recognition of exons may help explain the 300-nucleotide-length maximum of vertebrate internal exons, the mechanism whereby the splicing machinery ignores cryptic sites within introns, the mechanism whereby exon skipping is normally avoided, and the phenotypes of 5' splice site mutations that inhibit splicing of neighboring introns.

scholarly journals An Apparent Pseudo-Exon Acts both as an Alternative Exon That Leads to Nonsense-Mediated Decay and as a Zero-Length Exon

2006 ◽  
Vol 26 (6) ◽  
pp. 2237-2246 ◽  
Author(s):  
Sushma-Nagaraja Grellscheid ◽  
Christopher W. J. Smith
Keyword(s):  
Splice Site ◽  
Alternative Exon ◽  
Splice Sites ◽  
Exon 2 ◽  
Nonsense Mediated Decay ◽  
Exon Splicing ◽  
Exon 4 ◽  
Cryptic Splice Sites

ABSTRACT Pseudo-exons are intronic sequences that are flanked by apparent consensus splice sites but that are not observed in spliced mRNAs. Pseudo-exons are often difficult to activate by mutation and have typically been viewed as a conceptual challenge to our understanding of how the spliceosome discriminates between authentic and cryptic splice sites. We have analyzed an apparent pseudo-exon located downstream of mutually exclusive exons 2 and 3 of the rat α-tropomyosin (TM) gene. The TM pseudo-exon is conserved among mammals and has a conserved profile of predicted splicing enhancers and silencers that is more typical of a genuine exon than a pseudo-exon. Splicing of the pseudo-exon is fully activated for splicing to exon 3 by a number of simple mutations. Splicing of the pseudo-exon to exon 3 is predicted to lead to nonsense-mediated decay (NMD). In contrast, when “prespliced” to exon 2 it follows a “zero length exon” splicing pathway in which a newly generated 5′ splice site at the junction with exon 2 is spliced to exon 4. We propose that a subset of apparent pseudo-exons, as exemplified here, are actually authentic alternative exons whose inclusion leads to NMD.

scholarly journals A Splice Site Mutant of Maize Activates Cryptic Splice Sites, Elicits Intron Inclusion and Exon Exclusion, and Permits Branch Point Elucidation

1999 ◽  
Vol 121 (2) ◽  
pp. 411-418 ◽  
Author(s):  
Shailesh Lal ◽  
Jae-Hyuk Choi ◽  
Janine R. Shaw ◽  
L. Curtis Hannah
Keyword(s):  
Branch Point ◽  
Splice Site ◽  
Splice Sites ◽  
Cryptic Splice Sites

scholarly journals Role of the 3′ Splice Site in U12-Dependent Intron Splicing

2001 ◽  
Vol 21 (6) ◽  
pp. 1942-1952 ◽  
Author(s):  
Rosemary C. Dietrich ◽  
Marian J. Peris ◽  
Andrew S. Seyboldt ◽  
Richard A. Padgett
Keyword(s):  
Splice Site ◽  
Site Selection ◽  
Intron Splicing ◽  
Splice Sites ◽  
Splice Site Selection ◽  
Branch Site ◽  
Site Function

ABSTRACT U12-dependent introns containing alterations of the 3′ splice site AC dinucleotide or alterations in the spacing between the branch site and the 3′ splice site were examined for their effects on splice site selection in vivo and in vitro. Using an intron with a 5′ splice site AU dinucleotide, any nucleotide could serve as the 3′-terminal nucleotide, although a C residue was most active, while a U residue was least active. The penultimate A residue, by contrast, was essential for 3′ splice site function. A branch site-to-3′ splice site spacing of less than 10 or more than 20 nucleotides strongly activated alternative 3′ splice sites. A strong preference for a spacing of about 12 nucleotides was observed. The combined in vivo and in vitro results suggest that the branch site is recognized in the absence of an active 3′ splice site but that formation of the prespliceosomal complex A requires an active 3′ splice site. Furthermore, the U12-type spliceosome appears to be unable to scan for a distal 3′ splice site.

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