Methods for modulating RNA splicing
In one aspect, described herein is an intronic recognition element for splicing modifier (iREMS) that can be recognized by a compound provided herein. In another aspect, described herein are methods for modulating the amount of a product of a gene, wherein a precursor RNA transcript transcribed from the gene contains an intronic REMS, and the methods utilizing a compound described herein. More particularly, described herein are methods for modulating the amount of an RNA transcript or protein product encoded by a gene, wherein a precursor RNA transcript transcribed from the gene comprises an intronic REMS, and the methods utilizing a compound described herein. In another aspect, provided herein are artificial gene constructs comprising an intronic REMS, and uses of those artificial gene constructs to modulate protein production. In another aspect, provided herein are methods for altering endogenous genes to comprise an intronic REMS, and the use of a compound described herein to modulate protein produced from such altered endogenous genes.
1. A method for producing a mature mRNA transcript comprising an iExon from a pre-mRNA transcript, the method comprising contacting the pre-mRNA transcript with a compound, wherein the pre-mRNA transcript comprises two exons and an intron, wherein one exon is upstream of the intron and the other exon is downstream of the intron, wherein the intron comprises in 5′ to 3′ order: a first 5′ splice site, a first branch point, a first 3′ splice site, an endogenous intronic recognition element for splicing modifier (iREMS), a second branch point, and a second 3′ splice site, wherein the iREMS comprises an RNA sequence NNGAgurngn (SEQ ID NO: 1), wherein r is adenine or guanine and n or N is any nucleotide, wherein the pre-mRNA transcript is a pre-mRNA transcript of a gene that is selected from ABCB8, ABCC3, ADAM17, ADCY3, AGPAT4, ANKRA2, ANXA11, APIP, APPL2, ARHGAP1, ARL15, ASAP1, ATAD2B, ATXN1, BECN1, BHMT2, BICD1, BTN3A1, C11orf30, C11orf73, C12orf4, C14orf132, C8orf44, C8orf44-SGK3, C8orf88, CASC3, CASP7, CCDC122, CDH13, CECR7, CENPI, CEP112, CEP192, CHEK1, CMAHP, COPS7B, CPSF4, CRISPLD2, CRYBG3, CSNK1E, CSNK1G1, DCAF17, DCUN1D4, DDX42, DENND1A, DENND5A, DGKA, DHFR, DIAPH3, DNAJC13, DNMBP, DOCK1, DYRK1A, EIF2B3, ENAH, EP300, ERC1, ERLIN2, ERRFI1, EVC, FAF1, FAIM, FAM126A, FAM13A, FAM162A, FAM174A, FBN2, FER, FHOD3, FOCAD, GALC, GCFC2, GGACT, GLCE, GOLGA4, GOLGB1, GPSM2, GULP1, GXYLT1, HDX, HLTF, HMGA2, HNMT, HSD17B12, HSD17B4, HTT, IFT57, IVD, KDM6A, KIAA1524, KIAA1715, LETM2, LOC400927, LRRC42, LUC7L3, LYRM1, MB21D2, MCM10, MED13L, MEDAG, MEMO1, MFN2, MMS19, MRPL45, MRPS28, MTERF3, MYCBP2, MYLK, MYOF, NGF, NREP, NSUN4, NT5C2, OSMR, OXCT1, PAPD4, PCM1, PDE7A, PDS5B, PDXDC1, PIGN, PIK3CD, PIK3R1, PIKFYVE, PITPNB, PLEKHA1, PLSCR1, PMS1, POMT2, PPARG, PPIP5K2, PPP1R26, PRPF31, PRSS23, PSMA4, PXK, RAF1, RAPGEF1, RARS2, RBKS, RERE, RFWD2, RPA1, RPS10, SAMD4A, SAR1A, SCO1, SEC24A, SENP6, SERGEF, SGK3, SLC12A2, SLC25A17, SLC44A2, SMYD3, SNAP23, SNHG16, SNX7, SOS2, SPATA5, SPIDR, SPRYD7, SRGAP1, SRRM1, STAT1, STXBP6, SUPT20H, TAF2, TASP1, TBC1D15, TCF12, TCF4, TIAM1, TJP2, TMC3, TMEM214, TNRC6A, TNS3, TOE1, TRAF3, TSPAN2, TTC7B, TYW5, UBAP2L, URGCP, VAV2, WDR27, WDR37, WDR91, WNK1, XRN2, ZCCHC8, ZFP82, ZNF138, ZNF232 or ZNF37BP;
and wherein the compound is selected from 2-(4,6-dimethylpyrazolo[1,5-a]pyrazin-2-yl)-7-(4-methylpiperazin-1-yl)-4H-pyrido[1,2-a]pyrimidin-4-one, 2-(4,6-dimethylpyrazolo[1,5-a]pyrazin-2-yl)-7-(1-ethylpiperidin-4-yl)-9-methyl-4H-pyrido[1,2-a]pyrimidin-4-one, and 2-(4-ethyl-6-methylpyrazolo[1,5-a]pyrazin-2-yl)-9-methyl-7-(1-methylpiperidin-4-yl)-4H-pyrido[1,2-a]pyrimidin-4-one or a salt thereof.
2. The method of claim 1 , wherein
the RNA sequence NNGAgurngn (SEQ ID NO: 1) is NNGAguragu (SEQ ID NO: 3862), wherein r is adenine or guanine and N is any nucleotide.
3. The method of claim 1 , wherein
the RNA sequence NNGAgurngn (SEQ ID NO: 1) is selected from the group consisting of ANGAgurngn (SEQ ID NO: 29), CNGAgurngn (SEQ ID NO: 35), GNGAgurngn (SEQ ID NO: 41), UNGAgurngn (SEQ ID NO: 47), NAGAgurngn (SEQ ID NO: 30), NCGAgurngn (SEQ ID NO: 36), NGGAgurngn (SEQ ID NO: 42), NUGAgurngn (SEQ ID NO: 48), AAGAgurngn (SEQ ID NO: 31), ACGAgurngn (SEQ ID NO: 37), AGGAgurngn (SEQ ID NO: 43), AUGAgurngn (SEQ ID NO: 49), CAGAgurngn (SEQ ID NO: 32), CCGAgurngn (SEQ ID NO: 38), CGGAgurngn (SEQ ID NO: 44), CUGAgurngn (SEQ ID NO: 50), GAGAgurngn (SEQ ID NO: 33), GCGAgurngn (SEQ ID NO: 39), GGGAgurngn (SEQ ID NO: 45), GUGAgurngn (SEQ ID NO: 51), UAGAgurngn (SEQ ID NO: 34), UCGAgurngn (SEQ ID NO: 40), UGGAgurngn (SEQ ID NO: 46) and UUGAgurngn (SEQ ID NO: 52), wherein r is adenine or guanine and n or N is any nucleotide.
4. The method of claim 1 , wherein the pre-mRNA transcript is in a cell or a lysate of the cell and the method comprises contacting the cell or cell lysate with the compound.
5. The method of claim 2 , wherein the RNA sequence NNGAguragu (SEQ ID NO: 3862) is selected from the group consisting of ANGAguragu (SEQ ID NO: 437), CNGAguragu (SEQ ID NO: 443), GNGAguragu (SEQ ID NO: 449), UNGAguragu (SEQ ID NO: 455), NAGAguragu (SEQ ID NO: 438), NCGAguragu (SEQ ID NO: 444), NGGAguragu (SEQ ID NO: 450), NUGAguragu (SEQ ID NO: 456), AAGAguragu (SEQ ID NO: 439), ACGAguragu (SEQ ID NO: 445), AGGAguragu (SEQ ID NO: 451), AUGAguragu (SEQ ID NO: 457), CAGAguragu (SEQ ID NO: 440), CCGAguragu (SEQ ID NO: 446), CGGAguragu (SEQ ID NO: 452), CUGAguragu (SEQ ID NO: 458), GAGAguragu (SEQ ID NO: 441), GCGAguragu (SEQ ID NO: 447), GGGAguragu (SEQ ID NO: 453), GUGAguragu (SEQ ID NO: 459), UAGAguragu (SEQ ID NO: 442), UCGAguragu (SEQ ID NO: 448), UGGAguragu (SEQ ID NO: 454) and UUGAguragu (SEQ ID NO: 460), wherein r is adenine or guanine, and N is any nucleotide.
6. The method of claim 1 , wherein the compound is 2-(4,6-dimethylpyrazolo[1,5-a]pyrazin-2-yl)-7-(4-methylpiperazin-1-yl)-4H-pyrido[1,2-a]pyrimidin-4-one or a salt thereof.
7. The method of claim 1 , wherein the compound is 2-(4,6-dimethylpyrazolo[1,5-a]pyrazin-2-yl)-7-(1-ethylpiperidin-4-yl)-9-methyl-4H-pyrido[1,2-a]pyrimidin-4-one or a salt thereof.
8. The method of claim 1 , wherein the compound is 2-(4-ethyl-6-methylpyrazolo[1,5-a]pyrazin-2-yl)-9-methyl-7-(1-methylpiperidin-4-yl)-4H-pyrido[1,2-a]pyrimidin-4-one or a salt thereof.
9. The method of claim 1 , wherein the pre-mRNA transcript is a pre-mRNA transcript of a gene that is selected from ABCC3, ADCY3, AGPAT4, ANKRA2, ANXA11, APIP, APPL2, ARHGAP1, ASAP1, ATAD2B, ATXN1, BECN1, BHMT2, BICD1, BTN3A1, C11orf30, C11orf73, C14orf132, C8orf88, CASC3, CASP7, CCDC122, CDH13, CECR7, CEP112, CMAHP, CPSF4, CRISPLD2, CRYBG3, CSNK1G1, DCAF17, DCUN1D4, DDX42, DENND1A, DENND5A, DGKA, DIAPH3, DNAJC13, DNMBP, DOCK1, DYRK1A, EIF2B3, ERC1, ERLIN2, EVC, FAF1, FAIM, FAM126A, FAM13A, FAM174A, FBN2, FER, FHOD3, FOCAD, GALC, GCFC2, GGACT, GLCE, GOLGA4, GOLGB1, GXYLT1, HLTF, HMGA2, HNMT, HSD17B12, HSD17B4, HTT, IFT57, IVD, KIAA1524, KIAA1715, LUC7L3, LYRM1, MB21D2, MCM10, MED13L, MEDAG, MEMO1, MMS19, MRPL45, MRPS28, MTERF3, MYCBP2, MYOF, NREP, NSUN4, NT5C2, OSMR, OXCT1, PAPD4, PCM1, PDS5B, PIK3R1, PIKFYVE, PITPNB, PLSCR1, PMS1, PPIP5K2, PRSS23, PSMA4, PXK, RAF1, RARS2, RFWD2, RPA1, SAMD4A, SAR1A, SCO1, SEC24A, SERGEF, SMYD3, SNAP23, SNHG16, SNX7, SPATA5, SPIDR, SPRYD7, SRGAP1, STAT1, STXBP6, SUPT20H, TAF2, TBC1D15, TCF12, TJP2, TMEM214, TNS3, TOE1, TRAF3, TSPAN2, TYW5, URGCP, XRN2, and ZNF232.