The U1-like snRNA variants are ubiquitously expressed, and show features characteristic of spliceosomal snRNAs. (A) Northern blot analysis of snRNA variants in HeLa cells. The following probes were used: u1a (lane 1), u1a5 (lane 2), u1a6 (lane 3), u1a7 (lane 4), and 5s (lane 5). Positions of RNA size markers are indicated on the left. (B) RNA samples prepared from HeLa cells and a range of human tissues obtained from distinct developmental stages were analyzed by Northern blot analysis. The U1 snRNA variants detected by the specific oligonucleotide probes are indicated on the left and tissue types are indicated above the panels. RNA isolated from HeLa cells (lanes 1,10,11,19,20,28), adult tissues (lanes 2–9), 6-week embryonic tissue (lanes 12–18), and 12-week embryonic tissue (lanes 21–27). (C) Northern blot analysis of immunoprecipitation (IP) experiments. HeLa nuclear extract was subjected to IP using an anti-m₃G/m⁷G-cap monoclonal antibody (mAbH20; lane 1), an anti-snRNA-m₃G-cap polyclonal antiserum (r-R1131; lane 3), or an anti-Sm-protein monoclonal antibody (mAbY12; lane 5), respectively. Controls were included utilizing mouse immunoglobulin (mIgG, lanes 2,6) and normal rabbit serum (NRS, lane 4), respectively. Immunoprecipitated RNA was purified and analyzed by Northern blot analysis. U1 snRNA variants or 5S RNA detected by the specific oligonucleotide probes are indicated. (D) Northern blot analysis of electrophoretic mobility shift assays (EMSA) using nuclear extract preparations; nuclear extract (NE; lanes 1,3,5,7); nuclear RNA (RNA; lanes 2,4,6,8). The U1 snRNA variants detected by specific oligonucleotide probes are indicated above the panels. The position of RNA–protein complexes (snRNP) and free snRNA molecules (snRNA) are indicated.

U1A snRNA variants. (A) Alignment of the RNA sequences of the U1A, U1A4, U1A5, U1A6, and U1A7 snRNA variants. Nucleotides in U1A that are conserved in a variant snRNA are highlighted in green. Regions corresponding to important features within U1A snRNA are indicated by colored boxes beneath the alignment: 5′SS recognition motif (5′SS, blue); stem A (A, white); U1–70K protein binding site (U1–70K, yellow); U1-A protein binding site (U1-A, orange); Sm-binding site (Sm, gray). The numbers to the right refer to the number of nucleotides counted from the 5′ end of the individual snRNA. (B) Predicted secondary structures for the U1A, U1A5, U1A6, and U1A7 snRNA variants. Regions corresponding to important features within U1A snRNA are color coded as specified above. Identified variable positions are highlighted by black boxes. The regions of the snRNAs targeted by the sequence specific probes used for Northern blot analysis are indicated by black lines. (C) Interaction of U1A, U1A5, U1A6, and U1A7 snRNA variants with canonical GU dinucleotide 5′SS sequences. The 5′SS is shown as a sequence logo constructed from 252,159 canonical GU dinucleotide 5′SS sequences. The putative 5′SS recognition motifs of the U1A5, U1A6, and U1A7 snRNA variants are aligned to the 5′SS recognition motif of U1A snRNA. Nucleotides differing from the corresponding nucleotides in U1A snRNA are indicated in bold. The variable nucleotide position Y₈ in U1A6 snRNA is highlighted by a black box. (D) Distributions illustrating the interaction between the U1A, U1A5, U1A6, and U1A7 snRNA variants, respectively, and the above-specified set of canonical GU dinucleotide 5′SS sequences. 5′SSs have been grouped according to the number of possible base pairs.

Evolutionary analysis of the RNU1A5, RNU1A6, and RNU1A7 loci. (A) Loci corresponding to the human RNU1A5, RNU1A6, and RNU1A7 locus as identified in the chimpanzee (Pan troglodytes, P.t.), orangutan (Pongo pygmaeus, P.p.), rhesus macaque (Macaca mulatta, M.m.), common marmoset (Callithrix jacchus, C.j.), dog (Canis familiaris, C.f.), and cow (Bos Taurus, B.t.). The presence and similarity of an snRNA sequence at a specific locus is indicated by colored squares: similar to U1A5 snRNA (green), U1A6 snRNA (blue), U1A7 snRNA (yellow), or bona fide U1A snRNA (orange); presumably nonfunctional snRNA (dark gray); not detected (ND). Phylogenetic relationships are depicted according to the NCBI taxonomy database (not to scale). An asterisk next to a species name indicates that the loci were found in the NCBI trace archives. (B) Schematic representation of multiple alignments of the identified RNU1A5, RNU1A6, and RNU1A7 loci. The locations of the putative coding sequences are indicated by arrows above the alignment and are color coded as above. Flanking regions consist of 150 nucleotides upstream and downstream. The similarities of the sequences relative to the human sequence are indicated: identical nucleotides (boxes), mismatch or deletion (thin line), and insertion (thick line).