U5 small nuclear ribonucleoprotein: RNA structure analysis and ATP-dependent interaction with U4/U6.

To understand how the U5 small nuclear ribonucleoprotein (snRNP) interacts with other spliceosome components, its structure and binding to the U4/U6 snRNP were analyzed. The interaction of the U5 snRNP with the U4/U6 snRNP was studied by separating the snRNPs in HeLa cell nuclear extracts on glycerol gradients. A complex running at 25S and containing U4, U5, and U6 but not U1 or U2 snRNAs was identified. In contrast to results with native gel electrophoresis to separate snRNPs, this U4/U5/U6 snRNP complex requires ATP to assemble from the individual snRNPs. The structure of the U5 RNA within the U5 snRNP and the U4/5/6 snRNP complexes was then compared. Oligonucleotide-targeted RNase H digestion identified one RNA sequence in the U5 snRNP capable of base pairing to other nucleic acid sequences. Chemical modification experiments identified this sequence as well as two other U5 RNA sequences as accessible to modification within the U5 RNP. One of these regions is a large loop in the U5 RNA secondary structure whose sequence is conserved from Saccharomyces cerevisiae to humans. Interestingly, no differences in modification of free U5 snRNP as compared to U5 in the U4/U5/U6 snRNP complex were observed, suggesting that recognition of specific RNA sequences in the U5 snRNP is not required for U4/U5/U6 snRNP assembly.     

A small RNA of Mycoplasma capricolum that resembles eukaryotic U6 small nuclear RNA.

Mycoplasma capricolum, a parasitic prokaryote, contains several small stable RNAs, besides rRNAs and tRNAs. One of them, designated MCS4 RNA (125 nucleotides in length), has been isolated and sequenced. This RNA is abundant in the cell, and is encoded by two genes. Unexpectedly, MCS4 RNA has been found to reveal extensive sequence similarity to eukaryotic U6 snRNAs. This finding suggests that MCS4 and U6 snRNAs are derived from a common ancestral RNA that has existed before the divergence of prokaryotes and eukaryotes.     

U4 small nuclear RNA pseudogenes from rat genome have common truncated 3'-ends

Four U4 RNA pseudogenes were isolated and characterized from a rat genomic bank. The four pseudogenes contained sequences completely homologous to U4 RNA from nucleotides 1 to 67 and had common truncated 3'-ends. Three of the four pseudogenes were flanked by 14 to 18 nucleotide-long direct repeats. The structural features of these four U4 RNA pseudogenes are consistent with the hypothesis that these pseudogenes arose by RNA self-primed complementary DNA synthesis and integration into the genome (Van Arsdell et al., Cell 26:11-17, 1981).     

Primary and secondary structure of U8 small nuclear RNA

U8 small nuclear RNA is a new, capped, 140 nucleotides long RNA species found in Novikoff hepatoma cells. Its sequence is: m3GpppAmUmCGUCAGGA GGUUAAUCCU UACCUGUCCC UCCUUUCGGA GGGCAGAUAG AAAAUGAUGA UUGGAGCUUG CAUGAUCUGC UGAUUAUAGC AUUUCCGUGU AAUCAGGACC UGACAACAUC CUGAUUGCUU CUAUCUGAUUOH. This RNA is present in approximately 25,000 copies/cell, and it is enriched in nucleolar preparations. Like U1, U2, U4/U6, and U5 RNAs, U8 RNA was also present as a ribonucleoprotein associated with the Sm antigen. The rat U8 RNA was highly homologous (greater than 90%) to a recently characterized 5.4 S RNA from mouse cells infected with spleen focus-forming virus (Kato, N., and Harada, F. (1984) Biochim. Biophys. Acta, 782, 127-131). In addition to the U8 RNA, three other U small nuclear RNAs were found in anti-Sm antibody immunoprecipitates from labeled rat and HeLa cells. Each of these contained a m3GpppAm cap structure; their apparent chain lengths were 60, 130, and 65 nucleotides. These U small nuclear RNAs are designated U7, U9, and U10 RNAs, respectively.     

Loop I of U1 small nuclear RNA is the only essential RNA sequence for binding of specific U1 small nuclear ribonucleoprotein particle proteins.

The binding of the U1 small nuclear ribonucleoprotein (snRNP)-specific proteins C, A, and 70K to U1 small nuclear RNA (snRNA) was analyzed. Assembly of U1 snRNAs from bean and soybean and a set of mutant Xenopus U1 snRNAs into U1 snRNPs in Xenopus egg extracts was studied. The ability to bind proteins was analyzed by immunoprecipitation with monospecific antibodies and by a protein-sequestering assay. The only sequence essential for binding of the U1-specific proteins was the conserved loop sequence in the 5' hairpin of U1. Further analysis suggested that protein C binds directly to the loop and that the assembly of proteins A and 70K into the RNP requires mainly protein-protein interactions. Protein C apparently recognizes a specific RNA sequence rather than a secondary structural element in the RNA.     

Evolutionary origin of the U6 small nuclear RNA intron.

U6 is the most conserved of the five small nuclear RNAs known to participate in pre-mRNA splicing. In the fission yeast Schizosaccharomyces pombe, the single-copy gene encoding this RNA is itself interrupted by an intron (T. Tani and Y. Ohshima, Nature (London) 337:87-90, 1989). Here we report analysis of the U6 genes from all four Schizosaccharomyces species, revealing that each is interrupted at an identical position by a homologous intron; in other groups, including ascomycete and basidiomycete fungi, as well as more distantly related organisms, the U6 gene is colinear with the RNA. The most parsimonious interpretation of our data is that the ancestral U6 gene did not contain an intron, but rather, it was acquired via a single relatively recent insertional event.