The immunolocalization of small nuclear ribonucleoprotein particles in testicular cells during the cycle of the seminiferous epithelium of the adult rat

The objective of this study was to determine the cellular and subcellular distribution of small nuclear ribonucleoprotein particles (snRNPs) in the adult rat testis in relation to the different cell types at the various stages of the cycle of the seminiferous epithelium. The distribution of snRNPs in the nucleus and cytoplasm of germ cells was quantitated in an attempt to correlate RNA processing with morphological and functional changes occurring during the development of these cells. Light-microscopic immunoperoxidase staining of rat testes with polyclonal anti-Sm and monoclonal anti-Y12 antibodies localized spliceosome snRNPs in the nuclei and cytoplasm of germ cells up to step 10 spermatids. Nuclear staining was intense in Sertoli cells, spermatogonia, spermatocytes, and in the early steps of round spermatid development. Although comparatively weaker, cytoplasmic staining for snRNPs was strongest in mid and late pachytene spermatocytes and early round spermatids. Quantitative electron-microscopic immunogold labeling of Lowicryl embedded testicular sections confirmed the light-microscopic observations but additionally showed that the snRNP content peaked in the cytoplasm of midpachytene spermatocytes and in the nuclei of late pachytene spermatocytes. The immunogold label tended to aggregate into distinct loci over the nuclear chromatin. The chromatoid body of spermatids and spermatocytes and the finely granular material in the interstices of mitochondrial aggregates of spermatocytes were found to be additional sites of snRNP localization and were intensely labeled. This colocalization suggests that these dense cytoplasmic structures may be functionally related. Anti-U1 snRNP antibodies applied to frozen sections showed the same LM localization pattern as spliceosome snRNPs. Anti-U3 snRNP antibodies applied to frozen sections stained nucleoli of germ cells where pre-rRNA is spliced.     

U2 as well as U1 small nuclear ribonucleoproteins are involved in premessenger RNA splicing

Two different experimental approaches have provided evidence that both U2 and U1 snRNPs function in pre-mRNA splicing. When the U2 snRNPs in a nuclear extract are selectively degraded using ribonuclease H and either of two deoxyoligonucleotides complementary to U2 RNA, splicing activity is abolished. Mixing an extract in which U2 has been degraded with one in which U1 has been degraded recovers activity. Use of anti-(U2)RNP autoantibodies demonstrates that U2 snRNPs associate with the precursor RNA during in vitro splicing. At 60 min, but not at 0 min, into the reaction intron fragments that include the branch-point sequence are immunoprecipitated by anti-(U2)RNP. At all times, U1 snRNPs bind the 5' splice site of the pre-mRNA. Possible interactions of the U2 snRNP with the U1 snRNP and with the pre-mRNA during splicing are considered.     

The differential interaction of snRNPs with pre-mRNA reveals splicing kinetics in living cells

Precursor messenger RNA (pre-mRNA) splicing is catalyzed by the spliceosome, a large ribonucleoprotein (RNP) complex composed of five small nuclear RNP particles (snRNPs) and additional proteins. Using live cell imaging of GFP-tagged snRNP components expressed at endogenous levels, we examined how the spliceosome assembles in vivo. A comprehensive analysis of snRNP dynamics in the cell nucleus enabled us to determine snRNP diffusion throughout the nucleoplasm as well as the interaction rates of individual snRNPs with pre-mRNA. Core components of the spliceosome, U2 and U5 snRNPs, associated with pre-mRNA for 15-30 s, indicating that splicing is accomplished within this time period. Additionally, binding of U1 and U4/U6 snRNPs with pre-mRNA occurred within seconds, indicating that the interaction of individual snRNPs with pre-mRNA is distinct. These results are consistent with the predictions of the step-wise model of spliceosome assembly and provide an estimate on the rate of splicing in human cells.     

The structure of mammalian small nuclear ribonucleoproteins. Identification of multiple protein components reactive with anti-(U1)ribonucleoprotein and anti-Sm autoantibodies

The Sm small nuclear ribonucleoproteins (snRNPs) from mammalian cells have been characterized as containing U1, U2, U4, U5, and U6 RNA associated with some subset of at least 10 distinct polypeptides (called 68K, A, A', B, B', C, D, E, F, and G) that range in molecular weight from 68,000 to 11,000. Whereas this entire collection of snRNP particles is precipitated by patient anti-Sm autoantibodies, anti-(U1)RNP autoantibodies specifically recognize U1 snRNPs. Here, we have performed immunoblots using the sera from 29 patients and a mouse anti-Sm monoclonal antibody to identify which HeLa cell snRNP proteins carry anti-Sm or anti-(U1)RNP antigenic determinants. Strikingly, every serum surveyed, as well as the monoclonal antibody, recognizes determinants on two or more snRNP protein components. The three proteins, 68K, A, and C, that uniquely fractionate with U1 snRNPs are specifically reactive with anti-(U1)RNP sera in blots. Anti-Sm patient sera and the mouse monoclonal antibody react with proteins B, B', D, and sometimes E, one or more of which must be present on all Sm snRNPs. The blot results combined with data obtained from a refined 32P-labeled RNA immunoprecipitation assay reveal that, in our collection of the sera from 29 patients, anti-Sm rarely exists in the absence of equal or higher titers of anti-(U1)RNP; moreover, (U1)RNP sera often contain detectable levels of anti-Sm. Our findings further define the protein composition of the Sm snRNPs and raise intriguing questions concerning the relatedness of snRNP polypeptides and the mechanism of autoantibody induction.     

Antisense probes containing 2-aminoadenosine allow efficient depletion of U5 snRNP from HeLa splicing extracts.

RNA duplexes containing the modified base 2-amino-adenine in place of adenine are stabilized through the formation of three hydrogen bonds in 2-amino A.U base pairs. Antisense 2'-O-alkyloligoribonucleotide probes incorporating 2-aminoadenosine are thus able to efficiently affinity select RNP particles which are otherwise inaccessible. This has allowed the efficient and specific depletion of U5 snRNP from HeLa cell nuclear splicing extracts. U5 snRNP is shown to be essential for spliceosome assembly and for both steps of pre-mRNA splicing. The absence of U5 snRNP prevents the stable association of U4/U6 but not U1 and U2 snRNPs with pre-mRNA.     

Commitment of yeast pre-mRNA to the splicing pathway requires a novel U1 small nuclear ribonucleoprotein polypeptide, Prp39p.

The binding of a U1 small nuclear ribonucleoprotein (snRNP) particle to the 5' splice site region of a pre-mRNA is a primary step of intron recognition. In this report, we identify a novel 75-kDa polypeptide of Saccharomyces cerevisiae, Prp39p, necessary for the stable interaction of mRNA precursors with the snRNP components of the pre-mRNA splicing machinery. In vivo, temperature inactivation or metabolic depletion of Prp39p blocks pre-mRNA splicing and causes growth arrest. Analyses of cell extracts reveal a specific and dramatic increase in the electrophoretic mobility of the U1 snRNP particle upon Prp39p depletion and demonstrate that extracts deficient in Prp39p activity are unable to form either the CC1 or CC2 commitment complex band characteristic of productive U1 snRNP/pre-mRNA association. Immunological studies establish that Prp39p is uniquely associated with the U1 snRNP and is recruited with the U1 snRNP into splicing complexes. On the basis of these and related observations, we propose that Prp39p functions, at least in part, prior to stable branch point recognition by the U1 snRNP particle to facilitate or stabilize the U1 snRNP/5' splice site interaction.     

Human U1-70K ribonucleoprotein antigen gene: organization, nucleotide sequence, and mapping to locus 19q13.3

We have isolated and sequenced the gene encoding the human U1-70K snRNP protein. U1-70K is an RNA-binding protein that is a specific component of the U1 small nuclear ribonucleoprotein complex (snRNP) and constitutes the major anti-(U1) RNP autoimmune antigen. We have mapped the U1-70K gene to the distal portion of chromosome 19, at band q13.3. The gene is greater than 44 kb in size and consists of 11 exons. The general structure of the gene has been completely conserved during vertebrate evolution and accounts for the production of several different U1-70K mRNA species by alternative pre-mRNA splicing. Comparison of the predicted amino acid sequences of animal U1-70K proteins reveals a high degree of conservation, particularly in the region of the RNP consensus domain. Even more striking is the complete conservation of the nucleotide sequence of an alternative included/excluded exon containing an in-frame translational termination codon. This conservation also includes significant portions of the downstream intervening sequence. This extraordinary conservation at the nucleotide sequence level suggests that alternative splicing of this exon serves an important function, perhaps in regulating the production of functional U1-70K protein.     

Ribonucleoprotein complex formation during pre-mRNA splicing in vitro.

The ribonucleoprotein (RNP) structures of the pre-mRNA and RNA processing products generated during in vitro splicing of an SP6/beta-globin pre-mRNA were characterized by sucrose gradient sedimentation analysis. Early, during the initial lag phase of the splicing reaction, the pre-mRNA sedimented heterogeneously but was detected in both 40S and 60S RNP complexes. An RNA substrate lacking a 3' splice site consensus sequence was not assembled into the 60S RNP complex. The two splicing intermediates, the first exon RNA species and an RNA species containing the intron and the second exon in a lariat configuration (IVS1-exon 2 RNA species), were found exclusively in a 60S RNP complex. These two splicing intermediates cosedimented under a variety of conditions, indicating that they are contained in the same RNP complex. The products of the splicing reaction, accurately spliced RNA and the excised IVS1 lariat RNA species, are released from the 60S RNP complex and detected in smaller RNP complexes. Sequence-specific RNA-factor interactions within these RNP complexes were evidenced by the preferential protection of the pre-mRNA branch point from RNase A digestion and protection of the 2'-5' phosphodiester bond of the lariat RNA species from enzymatic debranching. The various RNP complexes were further characterized and could be distinguished by immunoprecipitation with anti-Sm and anti-(U1)RNP antibodies.     

The 5'' end domain of U2 snRNA is required to establish the interaction of U2 snRNP with U2 auxiliary factor(s) during mammalian spliceosome assembly.

Stable association of U2 snRNP with the branchpoint sequence of mammalian pre-mRNAs requires binding of a non-snRNP protein to the polypyrimidine tract. In order to determine how U2 snRNP contacts this protein, we have used an RNA containing the consensus 5' and the (Py)n-AG 3' splice sites but lacking the branchpoint sequence so as to prevent direct U2 snRNA base pairing to the branchpoint. Different approaches including electrophoretic separation of RNP complexes formed in nuclear extracts, RNase T1 protection immunoprecipitation assays with antibodies against snRNPs and UV cross-linking experiments coupled to immunoprecipitations allowed us to demonstrate that at least three splicing factors contact this RNA at 0 degree C without ATP. As expected, U1 snRNP interacts with the region comprising the 5' splice site. A protein of approximately 65,000 molecular weight recognizes the RNA specifically at the 5' boundary of the polypyrimidine tract. It could be either the U2 auxiliary factor (U2AF) (Zamore and Green (1989) PNAS 86, 9243-9247), the polypyrimidine tract binding protein (pPTB) (Garcia-Blanco et al. (1989) Genes and Dev. 3, 1874-1886) or a mixture of both. U2 snRNP also contacts the RNA in a way depending on p65 binding, thereby further arguing that the latter may correspond to the previously characterized U2AF and pPTB. Cleavage of U2 snRNA sequence by a complementary oligonucleotide and RNase H led us to conclude that the 5' terminus of U2 snRNA is required to ensure the contact between U2 snRNP and p65 bound to the RNA. More importantly, this conclusion can be extended to authentic pre-mRNAs. When we have used a human beta-globin pre-mRNA instead of the above artificial substrate, RNA bound p65 became precipitable by anti-(U2) RNP and anti-Sm antibodies except when the 5' end of U2 snRNA was selectively cleaved.     

Autoantibodies to the U2 small nuclear ribonucleoprotein in a patient with scleroderma-polymyositis overlap syndrome

Autoantibodies directed against the U2 small nuclear ribonucleoprotein (snRNP) have been found in the serum of a patient with scleroderma-polymyositis overlap syndrome. This specificity, called anti-(U2)-RNP, is distinct from all previously described autoantibodies, including those that precipitate related snRNPs: anti-Sm antibodies, which react with the entire set of U1, U2, U4, U5, and U6 snRNPs, and anti-(U1)RNP antibodies, which recognize only U1 snRNPs. From HeLa cell extracts, anti-(U2)RNP immunoprecipitates predominantly one 32P-labeled RNA species, identified as U2 small nuclear RNA, and six [35S]methionine-labeled protein bands, A' (Mr = 32,000), B (Mr = 28,000), D (Mr = 16,000), E (Mr = 13,000), F (Mr = 12,000), and G (Mr = 11,000). Protein blot analysis reveals that the A' protein carries (U2)RNP antigenic determinant(s) and therefore represents a polypeptide unique to the U2 snRNP; the B protein associated with U2 snRNPs may also be unique. Like U1 and the other Sm snRNPs, U2 snRNPs occupy a nuclear, non-nucleolar location and are antigenically conserved from insects to man. An antibody specific for the U2 snRNP will be useful in deciphering the function of this particle.