Figure 1. Turning U7 snRNA into a splicing modulation tool. (a) The U7 snRNP binds, via the 5′ end of its U7 snRNA moiety, to the histone downstream element (HDE) that follows the histone pre‐mRNA 3′ processing site (arrow). Additional factors involved in the 3′ end processing reaction are the histone hairpin binding protein (HBP (also called stem‐loop binding protein, SLBP), a zinc finger protein (ZFP) of 100 kDa (ZFP100), and a heat‐labile factor (HLF) composed of various proteins involved in cleavage/polyadenylation of poly(A) + mRNAs. CPSF‐73 is the processing endonuclease. Five Sm proteins and the two Lsm proteins Lsm10 and Lsm11 bind to the wild‐type U7 Sm binding site, the sequence of which is indicated below the cartoon. Lsm11 is essential for 3′ end cleavage [ 2, 27]; (b) By replacing the wild‐type U7 Sm binding site with a consensus sequence derived from spliceosomal snRNAs, the resulting RNA assembles with the seven Sm proteins found in spliceosomal snRNAs. As a result, this U7 Sm OPT RNA accumulates more efficiently in the nucleoplasm and will no longer mediate histone pre‐mRNA cleavage, although it can still bind to histone pre‐mRNA and act as a competitive inhibitor for wild‐type U7 snRNPs. By further replacing the sequence binding to the HDE with one complementary to a particular target in a splicing substrate, it is possible to create U7 snRNAs capable of modulating specific splicing events [ 1, 2].

Figure 2. Exon‐skipping strategies using modified U7 Sm OPT snRNAs.   (a) Thalassemic mutations in the second intron of the human β‐globin gene that have been used to establish U7‐based exon skipping. These mutations create 5′SSs at positions 654, 705, or 745 of the second β‐globin intron. This activates a cryptic 3′SS upstream in the same intron, resulting in the inclusion of an aberrant exon containing an early stop codon and, therefore, in the loss of β‐globin protein production. The asterisks indicate potential branchpoints; the black‐shaded area represents part of an aberrant intron fused in frame to exon 2, ending with a premature stop codon.   (b) U7 Sm OPT snRNA‐based strategies that have been used successfully. Top: Single‐target constructs targeting 3′SS, 5′SS or exon‐internal sequences, preferably exonic splicing enhancers. Bottom left: Double‐target U7 snRNAs that base‐pair simultaneously to two regions upstream and downstream of the exon, and which presumably act by forcing the exon of interest into a looped structure. Bottom right: Bifunctional U7 snRNA targeting an exon‐internal sequence and additionally containing a binding sequence for the splicing silencing protein hnRNP A1. Figure modified from Ref. [ 1]. See the main text for references for the individual strategies. Adapted from Ref. [ 6];

Figure 3. Exon‐inclusion strategies using modified U7 Sm OPT snRNAs. The cartoon shows exons 6–8 of the human SMN2 gene. Compared to the SMN1 gene, one of two splicing enhancers (SE1 and 2) is altered by a C→U transition, resulting in frequent skipping of SMN2 exon 7. (a) Exon 7 inclusion (and production of full‐length SMN protein) can be stimulated by a U7 snRNA derivative targeting the 3′SS of exon 8 [ 13]; (b) Alternatively, a bifunctional U7 snRNA that binds to exon 7 and carries a splicing enhancer sequence at its 5′ end can strongly stimulate exon 7 inclusion by providing a binding site for a splicing stimulatory SR protein, e.g., ASF/SF2 [ 14]. As SMA patients have no or only nonfunctional SMN1 genes, and the disease is caused by motorneuron death due to insufficient SMN protein levels, these strategies represent excellent options for a gene repair therapy.

Figure 4. PCR‐based introduction of functional sequences into U7 SmOPT. (a) Map of the relevant region of pSP64‐U7 Sm OPT and outline of Protocol 1. A mutagenic primer covering the StuI site, the histone antisense region (light green), and the Sm‐binding site (light blue) of U7 Sm OPT RNA is used in a PCR reaction with the pSP + vector primer (that can also be used to sequence inserts) and pSP64‐U7 Sm OPT as template. The PCR product and pSP64‐U7 Sm OPT are cut with StuI and HindIII, and the appropriate fragments are re‐ligated to generate the plasmid containing the desired replacements in the original antisense region (asterisks); (b) DNA sequence of the plasmid region shown in panel (a), with the relevant features indicated.

Figure 5. Subcloning of a U7 cassette into lentiviral vector pWPTS. The entire U7 snRNA cassette, including the U7 promoter and 3′ flanking region is amplified by PCR with mutagenic primers containing SfuI and ClaI recognition sequences. After cleavage with these restriction enzymes, the fragment can be ligated into ClaI‐cut pWPTS‐GFP. Note that SfuI and ClaI produce compatible 5′ overhangs. The cassette can go into the vector in both orientations. The ClaI junction will always restore a single ClaI site in the vector. Because of an overlap of the ClaI site with a DAM methylation site, the vector DNA must be amplified in an E. coli dam ^– strain in order to be cleavable by ClaI.

Figure 6. Agarose gel analysis of PCR reactions. A negative photograph of a 2% agarose gel is shown. The lanes are labeled with the names of the resulting U7 constructs that have been used in Ref. [ 14]. The corresponding sequences of the mutagenic primers are listed in the text in the same order. The PCR conditions were identical to those in Protocol 1, except that the annealing temperature was adjusted to 45 °C. The expected size of the PCR products is 339 bp (316 bp for U7‐notail‐A). These bands were later excised and the PCR products isolated from the gel, prior to digestion with StuI and HindIII.