Nucleotide sequence and organization of full length human U4 RNA pseudogenes

Two loci encoding human U4 RNA, designated U4/7 and U4/14, have been isolated and sequenced. Both are pseudogenes in that their sequences do not match any identified human U4 RNA species perfectly. The U4/7 locus harbours a full-length pseudogene of 144 bp with eight base substitutions in the structural region. This pseudogene might be derived from a hitherto unidentified human U4 RNA gene. The second locus, U4/14, has a complex structure; the structural sequence of a U4 gene has apparently been integrated into an Alu sequence.     

Restoration of correct splicing of thalassemic beta-globin pre-mRNA by modified U1 snRNAs

The T-->G mutation at nucleotide 705 in the second intron of the beta-globin gene creates an aberrant 5' splice site and activates a 3' cryptic splice site upstream from the mutation. As a result, the IVS2-705 pre-mRNA is spliced via the aberrant splice sites leading to a deficiency of beta-globin mRNA and protein and to the genetic blood disorder thalassemia. We have shown previously that in cell culture models of thalassemia, aberrant splicing of beta-thalassemic IVS2-705 pre-mRNA was permanently corrected by a modified murine U7 snRNA that incorporated sequences antisense to the splice sites activated by the mutation. To explore the possibility of using other snRNAs as vectors for antisense sequences, U1 snRNA was modified in a similar manner. Replacement of the U1 9-nucleotide 5' splice site recognition sequence with nucleotides complementary to the aberrant 5' splice site failed to correct splicing of IVS2-705 pre-mRNA. In contrast, U1 snRNA targeted to the cryptic 3' splice site was effective. A hybrid with a modified U7 snRNA gene under the control of the U1 promoter and terminator sequences resulted in the highest levels of correction (up to 70%) in transiently and stably transfected target cells.     

Proximity of the U12 snRNA with both the 5' splice site and the branch point during early stages of spliceosome assembly

U12 snRNA is required for branch point recognition in the U12-dependent spliceosome. Using site-specific cross-linking, we have captured an unexpected interaction between the 5' end of the U12 snRNA and the -2 position upstream of the 5' splice site of P120 and SCN4a splicing substrates. The U12 snRNA nucleotides that contact the 5' exon are the same ones that form the catalytically important helix Ib with U6atac snRNA in the spliceosome catalytic core. However, the U12/5' exon interaction is transient, occurring prior to the entry of the U4atac/U6atac.U5 tri-snRNP to the spliceosome. This suggests that the helix Ib region of U12 snRNA is positioned near the 5' splice site early during spliceosome assembly and only later interacts with U6atac to form helix Ib. We also provide evidence that U12 snRNA can simultaneously interact with 5' exon sequences near 5' splice site and the branch point sequence, suggesting that the 5' splice site and branch point sequences are separated by <40 to 50 A in the complex A of the U12-dependent spliceosome. Thus, no major rearrangements are subsequently needed to position these sites for the first step of catalysis.