The role of the U2–U12 hybrid intron in alternative splicing of human JNK2 pre-mRNA

<p><b>Copyright information:</b></p><p>Taken from "Alternative splicing and bioinformatic analysis of human U12-type introns"</p><p></p><p>Nucleic Acids Research 2007;35(6):1833-1841.</p><p>Published online 1 Mar 2007</p><p>PMCID:PMC1874599.</p><p>© 2007 The Author(s)</p> Schematic diagram shows exons 5 to 7 of JNK2. Between alternative exon 6a and 6b is the U2–U12 hybrid intron (heavy line); mutually exclusive selection of either exon generates mRNA isoforms. The numbers indicate the exon or intron length in nucleotides. U2 and U12 splice sites are indicated by hatched and filled boxes, respectively. The triangle indicates an EcoRV restriction site in exon 6b. Below the diagram is the sequence of the Py tract of the hybrid intron. The minigene contains exons 5 to 8 of human JNK2, in which all introns except for the hybrid one are internally truncated (length indicated by the numbers). Transcription of the minigenes is driven by the CMV promoter. Translation start codon was in frame introduced in exon 5. Within the hybrid intron, the vertical line and grey boxes indicate the site for exon 6a/b swap and CU-rich sequences, respectively. The E6a and E6b control PCR products were amplified from the minigene, whereas the E6a and E6b transcripts represent the RT-PCR products that were amplified from the JNK transcripts. EcoRV digestion of the exon 6b-containing DNA fragment (472 bp) yielded two bands (294 and 178 bp). The modified minigenes are also depicted; the numbers of total and C/U residues of their polypyrimidine (Py) tract are listed. Analysis of the JNK transcripts of four cell lines. The first four lanes show the E6a and E6b control fragments with or without EcoRV digestion. RT-PCR DNA fragments amplified from the endogenous JNK2 transcripts were of 316 bp; EcoRV digestion of the exon 6b-containing product generates two nearly comigrating fragments (cut) of 171 and 145 bp. Percentage of E6a-containing transcripts [cut/(cut + uncut) × 100%] in four cell lines was measured from three independent experiments; average with standard deviation is indicated. HEK 293 cells were transfected with a minigene reporter as indicted. Total RNA was collected 24-h post-transfection and analyzed as in panel B. Shown on the gel are uncut PCR products (634 bp) and the larger digested fragment (463 bp). The graph represents absolute sequence complexity (Y-axis) of a JNK2 genomic segment containing the alternative exon 6a/b and the hybrid intron (X-axis) from eight vertebrates (human, chimp, dog, cow, rat, mouse, opossum and chicken). Conservation of the Py tracts is indicated as percentage. Model shows that mutually exclusive exon (hatched and squared boxes) selection of the JNK2 gene is driven by the U2–U12 intron (heavy line). The Py tract (grey box) of the hybrid intron dominates over that upstream of exon 6a, thus leading to exon 6b inclusion as the default pathway, particularly in non-neuronal cells. In neuronal cells, specific splicing activators, such as Nova, induce the use of exon 6a (,). On the other hand, since several YCAY elements (vertical lines) exist in the Py tract of the hybrid intron, YCAY-binding factors (such as Nova) may antagonize the activity of the ubiquitous CU-rich element-binding factors to reduce the utilization of exon 6b

Effects of single nucleotide polymorphisms of the human WDFY1 U12-type intron

<p><b>Copyright information:</b></p><p>Taken from "Alternative splicing and bioinformatic analysis of human U12-type introns"</p><p></p><p>Nucleic Acids Research 2007;35(6):1833-1841.</p><p>Published online 1 Mar 2007</p><p>PMCID:PMC1874599.</p><p>© 2007 The Author(s)</p> Schematic diagram shows exons 4 to 7 of the human WDFY1 gene. Nucleotide variations were found at residue 6 (+6) of the 5′SS consensus sequence and residue 14 (−14) upstream of the branch site. The minigenes used for the splicing assay contain a single mutation at either the 5′ + 6 or the B-4 position or dual mutations (5′Bdm) at both sites. The numbers indicate the exon or intron length in nucleotides. The minigene is composed of exons 4 to 7 with internally truncated introns (length indicated by the numbers). Translation start and stop codons are in frame at the 5′ end of exon 4 and 3′ end of exon 7, respectively. The use of the cryptic splice sites b1, b2, d, c yielded aberrant transcripts (see panel C). HEK 293 cells were transfected with a minigene reporter as indicted. Total RNA was collected 24-h post-transfection and subjected to RT-PCR analysis using P-labeled primers. The identity of the cDNA products is shown at the right; hatched boxes represent truncated exons. The bar graph shows the relative abundance of the major splicing products as a percentage; the data were obtained from two to three independent experiments. The ∼330-bp band (asterisk) included a variety of aberrantly spliced JNK products, none of which expressed dominantly, and therefore was tentatively ignored in the graph. A U12 snRNA expression vector was co-transfected with the wild-type or B-4CA WDFY1 minigene into HEK 293 cells. Splicing of the WDFY1 transcripts was examined as in panel C. The bar graph is as in panel C. Model shows alternative splicing of the WDFY1 U12-type intron induced by genetic mutations. A 5′SS mutation activates cryptic U2-type 5′SSs in exon 5 (upper panel). A branch-site consensus mutation promotes the use of cryptic 5′SSs in exon 5 by the U2-type spliceosome or cryptic 3′SSs in exon 6 by the U2-type spliceosome (middle panel). Bottom: A double mutant is spliced only by the U2-type spliceosome, yielding aberrant mRNA products (lower panel)

U12-type introns present in splicing factor genes may impact on gene expression

<p><b>Copyright information:</b></p><p>Taken from "Alternative splicing and bioinformatic analysis of human U12-type introns"</p><p></p><p>Nucleic Acids Research 2007;35(6):1833-1841.</p><p>Published online 1 Mar 2007</p><p>PMCID:PMC1874599.</p><p>© 2007 The Author(s)</p> When the level or activity of a splicing factor encoded by a U12-type intron-containing gene is altered due to defective U12-type intron splicing (grey lines), aberrant splicing of its target pre-mRNAs may thus be observed

Additive Promotion of Viral Internal Ribosome Entry Site-Mediated Translation by Far Upstream Element-Binding Protein 1 and an Enterovirus 71-Induced Cleavage Product

<div><p>The 5' untranslated region (5' UTR) of the enterovirus 71 (EV71) RNA genome contains an internal ribosome entry site (IRES) that is indispensable for viral protein translation. Due to the limited coding capacity of their RNA genomes, EV71 and other picornaviruses typically recruit host factors, known as IRES <i>trans</i>-acting factors (ITAFs), to mediate IRES-dependent translation. Here, we show that EV71 viral proteinase 2A is capable of cleaving far upstream element-binding protein 1 (FBP1), a positive ITAF that directly binds to the EV71 5' UTR linker region to promote viral IRES-driven translation. The cleavage occurs at the Gly-371 residue of FBP1 during the EV71 infection process, and this generates a functional cleavage product, FBP1^1-371. Interestingly, the cleavage product acts to promote viral IRES activity. Footprinting analysis and gel mobility shift assay results showed that FBP1^1-371 similarly binds to the EV71 5' UTR linker region, but at a different site from full-length FBP1; moreover, FBP1 and FBP1^1-371 were found to act additively to promote IRES-mediated translation and virus yield. Our findings expand the current understanding of virus-host interactions with regard to viral recruitment and modulation of ITAFs, and provide new insights into translational control during viral infection.</p></div

<i>In vitro</i> induction of FBP1 cleavage by EV71 viral proteinase 2A.

<p>(A) 10 μg of wild-type 2A^pro (2A) or mutant 2A^pro (2A^C110S), wild-type 3C^pro (3C) or mutant 3C^pro (3C^C147S) viral proteinases were added to RD cell lysates and incubated for 4 hours at 37°C. Cleavage of eIF4G and CstF-64 respectively served as positive controls for 2A^pro and 3C^pro activity. (B) [³⁵S] methionine-labeled FBP1 was incubated with purified EV71 2A^pro or mutant 2A^pro (2A^C110S) for 4 hours at varying doses, or for (C)15 minutes to 4 hours at a fixed dose of 5 μg at 37°C. Proteins were then separated by SDS-PAGE and analyzed by autoradiography. Cp-N and Cp-C: Cleavage products of FBP1.</p

FBP1 cleavage following EV71 infection is independent of cellular proteasome, lysosome, or caspase activities.

<p>(A) EV71-infected or mock-infected RD cells were treated with 20 μM MG132 or 20 mM NH₄Cl at 3 h.p.i. Cell extracts at the indicated time points were analyzed with anti-FBP1, anti-3D^pol and anti-actin antibodies. Viral 3D^pol was used as an indicator for viral replication. (B) Cells were treated with 20 μM QVD-OPh, and lysates from the indicated time points were analysed with anti-FBP1, anti-PARP, anti-3C and anti-actin antibodies. Detection of PARP and its cleavage product (PARP Cp) was used as a positive control for viral-induced caspase activity. Viral 3C protein was used as an indicator for virus infection, and actin served as a loading control. Cps: Cleavage products of FBP1.</p

Function, expression and subcellular localization of FBP1 in EV71-infected cells.

<p>(A) RD cells were transduced with lentivirus carrying shNC (control) or shFBP1. Knockdown efficiency of FBP1 was confirmed by immunoblotting with antibodies that recognize the N-terminal epitope (Ab-N) of FBP1. Signal intensities of immunoblotting were quantified by ImageJ software, and the ratios of FBP1 intensity to actin are shown at bottom. <i>In vitro</i> (B) IRES-dependent and (C) cap-dependent translation were performed using EV71 5′ UTR-FLuc monocistronic RNA. Reactions were incubated with shNC or shFBP1 RD cytoplasmic extracts in the presence or absence of 250 nM of recombinant FBP1, and (D) 50% of the translation reactant were subjected to immunoblotting. Luciferase activity exhibited by the reporter was monitored with a luminometer. Error bars represent the standard deviation from three experimental and three technical replicates. P value was determined by two-tailed Student’s t test (**, <i>p</i> < 0.01). (E) Proteins expressed by RD cells at 2–10 hours post-infection (h.p.i.) by EV71 were analyzed by immunoblot analysis using anti-FBP1, anti-3D^pol, and anti-actin antibodies. Cp: Cleavage product of FBP1. Viral RNA was extracted at 2–10 h.p.i. and analyzed by slot-blotting. (F) Proteins in the cytoplasmic and nuclear fractions taken from cells at 2–6 h.p.i. were analyzed by immunoblot analysis, using anti-FBP1, anti-Lamin A/C, anti-GAPDH, and anti-3D^pol antibodies. M: mock-infected cells; C: cytoplasmic fraction; N: nuclear fraction.</p

Additive effects of FBP1 and FBP1^1-371 on IRES-driven translation.

<p>(A) FBP1 and FBP1^1-371 impact on EV71 IRES activity <i>in vitro</i>. EV71 5′ UTR-FLuc RNA was translated with shFBP1-RD cytoplasmic extracts, in the presence of increasing amounts of recombinant FBP1 or FBP1^1-371. Reactions without FBP1 or FBP1^1-371 were used as controls. Luciferase activity exhibited by the reporter was monitored with a luminometer. (B) Additive effect of FBP1 and FBP1^1-371 on EV71 IRES-driven translation <i>in vitro</i>. Recombinant FBP1, FBP1^1-371 or a combination of FBP1 and FBP1^1-371, were respectively added with EV71 5′ UTR-FLuc RNA to shFBP1-RD cytoplasmic extracts. A reaction without FBP1 or FBP1^1-371 was used as a control. (C) Effects of FBP1 and FBP1^1-371 binding to the linker region on EV71 IRES-driven translation <i>in vitro</i>. Recombinant FBP1, FBP1^1-371 or a combination of FBP1 and FBP1^1-371, were respectively added to wild-type EV71 5′ IRES-FLuc RNA, FBP1^1-371 binding site mutant EV71-IRES-mB1-FLuc RNA, FBP1 binding site mutant EV71-IRES-mB2-FLuc RNA, or FBP1 and FBP1^1-371 binding site-double mutant EV71-IRES-mB1B2-FLuc RNA in shFBP1-RD cytoplasmic extracts. A reaction without FBP1 or FBP1^1-371 was used as a control. (D) EV71 replicon 3D^D330A (upper panel), which is defective in viral RNA replication, was used to determine the effects of FBP1 and FBP1(G371K) on viral protein translation. shFBP1-RD cells were transiently transfected with a vector control or plasmids expressing FLAG-tagged human FBP1 or FBP1(G371K) wobble mutant [FBP1^R and FBP1(G371K)^R], which are resistant to the targeting of shFBP1. At 48 hours post-transfection, the cells were subsequently transfected with EV71 replicon 3D^D330A RNA. Luciferase activity exhibited by the cells were then monitored using a luminometer at 6 hours after the second transfection. Relative amounts of transfected replicon RNA and protein expression levels in each experimental set were also tested. The experiments conducted in A-D were repeated three times, and each sample was prepared in triplicate, while the results were analyzed statistically by Student’s t-test. Error bar: standard deviation; *: <i>p</i> <0.05 and **: <i>p</i> <0.01. (E) shFBP1-RD cells were transiently transfected with a vector control or plasmids expressing FLAG-tagged FBP1^R or FBP1(G371K)^R. At 48 hours post-transfection, the cells were subsequently infected with EV71 at a m.o.i. of 40, and viral titers during the course of infection were titrated by plaque assays. Relative amounts of protein expression levels in each experimental set were also tested. The data were analyzed statistically by one-way ANOVA. Error bar: standard deviation; *: <i>p</i> <0.05 and **: <i>p</i> <0.01.</p

Confirmation of FBP1 cleavage at Gly-371 by EV71 2A^pro.

<p>(A) [³⁵S] methionine-labeled FBP1 and FBP1 fragments containing aa 1–371, 372–644, 1–443, 185–644, and 185–443 were treated (+) or untreated (-) with EV71 2A^pro (2A). (B) Schematic representation of FBP1, FBP1 fragments and the proposed primary cleavage site at Gly-371 (indicated by an arrow). The molecular masses of the corresponding cleavage products are also shown. (C) RD cells transfected with FLAG-HA dual-tagged FBP1 and mutant FBP1^G371K, the latter of which is resistant to 2A^pro cleavage, were infected with EV71. At 4, 6, 8 and 10 h.p.i., cell lysates were prepared and analyzed by immunoblotting with anti-FLAG, anti-HA, anti-EV71 3D^pol and anti-actin antibodies. Cleavage products containing the FLAG-tag or HA-tag are indicated as Cp. Cp-N and Cp-C: Cleavage products respectively containing the N-terminal or C-terminal region of FBP1.</p

Interactome Analysis of the NS1 Protein Encoded by Influenza A H1N1 Virus Reveals a Positive Regulatory Role of Host Protein PRP19 in Viral Replication

Influenza A virus, which can cause severe respiratory illnesses in infected individuals, is responsible for worldwide human pandemics. The NS1 protein encoded by this virus plays a crucial role in regulating the host antiviral response through various mechanisms. In addition, it has been reported that NS1 can modulate cellular pre-mRNA splicing events. To investigate the biological processes potentially affected by the NS1 protein in host cells, NS1-associated protein complexes in human cells were identified using coimmunoprecipitation combined with GeLC–MS/MS. By employing software to build biological process and protein–protein interaction networks, NS1-interacting cellular proteins were found to be related to RNA splicing/processing, cell cycle, and protein folding/targeting cellular processes. By monitoring spliced and unspliced RNAs of a reporter plasmid, we further validated that NS1 can interfere with cellular pre-mRNA splicing. One of the identified proteins, pre-mRNA-processing factor 19 (PRP19), was confirmed to interact with the NS1 protein in influenza A virus-infected cells. Importantly, depletion of PRP19 in host cells reduced replication of influenza A virus. In summary, the interactome of influenza A virus NS1 in host cells was comprehensively profiled, and our findings reveal a novel regulatory role for PRP19 in viral replication