Regulation of doublesex pre-mRNA processing occurs by 3'-splice site activation

Sex-specific alternative processing of the doublesex (dsx) pre-mRNA controls somatic sexual differentiation in Drosophila melanogaster. Processing in the female-specific pattern results from the utilization of an upstream 3'-terminal exon and requires the activities of both the transformer (tra) and transformer-2 (tra-2) genes. Use of the more downstream male-specific terminal exons does not require the activities of these genes and is thus considered the default dsx-processing pattern. Here, we used transient expression of dsx pre-mRNAs in the presence or absence of tra and tra-2 gene products in Drosophila tissue culture cells to investigate the molecular mechanism controlling this alternative RNA-processing decision. These studies reveal that female-specific processing of dsx pre-mRNA is controlled by tra and tra-2 through the positive regulation of female-specific alternative 3'-terminal exon use. Delineation of cis-acting sequences necessary for regulation shows that a 540-nucleotide region from within the female exon is both necessary and sufficient for regulation. In addition, utilization of the female-specific 3'-splice site (3'SS) is regulated independently of female-specific polyadenylation. Regulated polyadenylation was obtained only in the presence of splicing, suggesting that activation of female-specific exon use occurs by 3'SS activation.     

Alternative splicing in the neural cell adhesion molecule pre-mRNA: regulation of exon 18 skipping depends on the 5'-splice site

Two isoforms of the neural cell adhesion molecule (NCAM), termed NCAM-180 and NCAM-140, derive from a single gene via inclusion or exclusion of the penultimate exon 18 (E18). This alternative splicing event is tissue-specific and regulated during differentiation. To explore its structural basis, we have analyzed the pattern of spliced mRNA generated from transiently transfected minigenes construct containing this exon and portions of the adjacent introns and exons faithfully reproduces the differentiation state-dependent alternative splicing of the endogenous pre-mRNA. By systematic deletion and replacement analysis, we scanned the minigene for the presence of functionally important cis-elements. We identified two sequences that affected differentiation state-dependent regulation. One, the central part of E18, does not seem to contain a specific cis-element essential for proper splice site choice, because extending the deletion restored correctly regulated expression of the splicing products. In contrast, the 5'-splice site is an important element for regulation. Replacing it with a corresponding sequence from the alpha-globin gene resulted in constitutive use of the optional exon. When placed in the alpha-globin gene it did not promote alternative splicing. Instead, we observed a strongly decreased efficiency of splicing of the downstream intron in undifferentiated cells. This block of splicing was partially relieved after differentiation. The results are consistent with a model in which skipping of E18 is controlled in part at the associated 5'-splice site by trans-acting factors that undergo quantitative or qualitative changes during differentiation of N2a cells.     

The Prp18 protein stabilizes the interaction of both exons with the U5 snRNA during the second step of pre-mRNA splicing

Interaction of the ends of the exons with loop 1 of the U5 snRNA aligns the exons for ligation in the second step of pre-mRNA splicing. To study the effect of Prp18 on the exons' interactions, we analyzed the splicing of pre-mRNAs with random sequences in the exon bases at the splice junctions. The exon mutations had large effects on splicing in yeast with a Prp18 protein lacking its most conserved region, but not in wild-type yeast. Analysis of splicing kinetics demonstrated that only the second step was affected in vivo and in vitro, showing that Prp18 - and specifically its conserved region - plays a key role in stabilizing the interaction of the exons with the spliceosome at the time of exon joining. Superior exon sequences defined by the prp18 results accelerated the second step of splicing by wild-type spliceosomes with inefficient AT-AC pre-mRNAs, implying that normal exon interactions follow the rules we discerned for prp18 splicing. Our results show that As are preferred at the ends of both exons and support a revised model of the interactions of the exons with U5 in which the exons are arranged in a continuous double helix that facilitates the second reaction.     

U5 snRNP在前体mRNA剪接加工中的作用

前体mRNA的剪接是基因表达过程中的关键一步,发生在基因的转录之后与蛋白合成之前。在由前体mRNA剪接加工而形成成熟mRNA时,需要将转录本中的内含子切除,因为它会干扰基因的表达。前体mRNA的剪接发生在细胞核中,是在一个大的RNA与蛋白质的复合体即剪接体的催化下完成的。复合体中含有U1、U2、U5,二聚体形式的U4/U6小核RNA(snRNA)和一些小核核糖核蛋白(snRNP)。U5snRNP特异蛋白包括hPrp8,hSnu114(aGTPase),hBrr2(aDExH/Dboxhelicase)和Prp28等。Prp8构成剪接体的催化核心,hSnu114可避免剪接复合体过早的活化。因此,U5snRNP在剪接体聚集过程和前体mRNA的剪接反应中发挥重要作用。     

Characterization of U4 and U6 interactions with the 5' splice site using a S. cerevisiae in vitro trans-splicing system

Spliceosome assembly has been characterized as the ordered association of the snRNP particles U1, U2, and U4/U6.U5 onto pre-mRNA. We have used an in vitro trans-splicing/cross-linking system in Saccharomyces cerevisiae nuclear extracts to examine the first step of this process, 5' splice site recognition. This trans-splicing reaction has ATP, Mg(2+), and splice-site sequence requirements similar to those of cis-splicing reactions. Using this system, we identified and characterized a novel U4-5' splice site interaction that is ATP-dependent, but does not require the branch point, the 3' splice site, or the 5' end of the U1 snRNA. Additionally, we identified several ATP-dependent U6 cross-links at the 5' splice site, indicating that different regions of U6 sample it before a U6-5' splice site interaction is stabilized that persists through the first step of splicing. This work provides evidence for ATP-dependent U4/U6 association with the 5' splice site independent of ATP-mediated U2 association with the branch point. Furthermore, it defines specific nucleotides in U4 and U6 that interact with the 5' splice site at this early stage, even in the absence of base-pairing with the U1 snRNA.     

Suppressors of a U4 snRNA mutation define a novel U6 snRNP protein with RNA-binding motifs

U4 and U6 small nuclear RNAs are associated by an extensive base-pairing interaction that must be disrupted and reformed with each round of splicing. U4 mutations within the U4/U6 interaction domain destabilize the complex in vitro and cause a cold-sensitive phenotype in vivo. Restabilization of the U4/U6 helix by dominant (gain-of-function), compensatory mutations in U6 results in wild-type growth. Cold-insensitive growth can also be restored by two classes of recessive (loss-of-function) suppressors: (1) mutations in PRP24, which we show to be a U6-specific binding protein of the RNP-consensus family; and (2) mutations in U6, which lie outside the interaction domain and identify putative PRP24-binding sites. Destabilization of the U4/U6 helix causes the accumulation of a PRP24/U4/U6 complex, which is undetectable in wild-type cells. The loss-of-function suppressor mutations inhibit the binding of PRP24 to U6, and thus presumably promote the release of PRP24 from the PRP24/U4/U6 complex and the reformation of the base-paired U4/U6 snRNP. We propose that the PRP24/U4/U6 complex is normally a highly transient intermediate in the spliceosome cycle and that PRP24 promotes the reannealing of U6 with U4.     

The Prp8 RNase H-like domain inhibits Brr2-mediated U4/U6 snRNA unwinding by blocking Brr2 loading onto the U4 snRNA

The spliceosomal RNA helicase Brr2 catalyzes unwinding of the U4/U6 snRNA duplex, an essential step for spliceosome catalytic activation. Brr2 is regulated in part by the spliceosomal Prp8 protein by an unknown mechanism. We demonstrate that the RNase H (RH) domain of yeast Prp8 binds U4/U6 small nuclear RNA (snRNA) with the single-stranded regions of U4 and U6 preceding U4/U6 stem I, contributing to its binding. Via cross-linking coupled with mass spectrometry, we identify RH domain residues that contact the U4/U6 snRNA. We further demonstrate that the same single-stranded region of U4 preceding U4/U6 stem I is recognized by Brr2, indicating that it translocates along U4 and first unwinds stem I of the U4/U6 duplex. Finally, we show that the RH domain of Prp8 interferes with U4/U6 unwinding by blocking Brr2''s interaction with the U4 snRNA. Our data reveal a novel mechanism whereby Prp8 negatively regulates Brr2 and potentially prevents premature U4/U6 unwinding during splicing. They also support the idea that the RH domain acts as a platform for the exchange of U6 snRNA for U1 at the 5′ splice site. Our results provide insights into the mechanism whereby Brr2 unwinds U4/U6 and show how this activity is potentially regulated prior to spliceosome activation.     

Structure of the Echinococcus multilocularis U1 snRNA gene repeat.

The gene encoding U1 snRNA in Echinococcus multilocularis has been cloned and sequenced. This gene is contained within a 1300-bp sequence which is tandemly repeated in the E. multilocularis genome. E. multilocularis U1 snRNA is 50-70% homologous to U1 snRNAs of other species. E. multilocularis U1 snRNA could assume a predicted secondary structure similar to that proposed for other U1 snRNAs, and appears shorter (157 bases) than the U1 snRNAs of higher eukaryotes (163-166 bases).     

A compensatory base change in human U2 snRNA can suppress a branch site mutation

We have developed an assay to test whether U2 snRNA can base-pair with the branch site during mammalian mRNA splicing. The beta 110 point mutation (GG----AG) within the first intron of human beta-globin generates a new 3' splice site that is preferentially used. We show here that use of the normal 3' splice site can be restored either by improving the match of a cryptic branch site to the branch site consensus or by introducing mutant U2 snRNAs with greater complementarity to the cryptic branch site. These data indicate that human U2 snRNA can form base pairs with the mRNA precursor; however, base pairing appears to be optional because some mammalian branch sites do not match the consensus.     

PRPF mutations are associated with generalized defects in spliceosome formation and pre-mRNA splicing in patients with retinitis pigmentosa

Proteins PRPF31, PRPF3 and PRPF8 (RP-PRPFs) are ubiquitously expressed components of the spliceosome, a macromolecular complex that processes nearly all pre-mRNAs. Although these spliceosomal proteins are conserved in eukaryotes and are essential for survival, heterozygous mutations in human RP-PRPF genes lead to retinitis pigmentosa, a hereditary disease restricted to the eye. Using cells from patients with 10 different mutations, we show that all clinically relevant RP-PRPF defects affect the stoichiometry of spliceosomal small nuclear RNAs (snRNAs), the protein composition of tri-small nuclear ribonucleoproteins and the kinetics of spliceosome assembly. These mutations cause inefficient splicing in vitro and affect constitutive splicing ex-vivo by impairing the removal of at least 9% of endogenously expressed introns. Alternative splicing choices are also affected when RP-PRPF defects are present. Furthermore, we show that the steady-state levels of snRNAs and processed pre-mRNAs are highest in the retina, indicating a particularly elevated splicing activity. Our results suggest a role for PRPFs defects in the etiology of PRPF-linked retinitis pigmentosa, which appears to be a truly systemic splicing disease. Although these mutations cause widespread and important splicing defects, they are likely tolerated by the majority of human tissues but are critical for retinal cell survival.     

mRNA-type introns in U6 small nuclear RNA genes: implications for the catalysis in pre-mRNA splicing

U6 small nuclear RNA is one of the spliceosomal RNAs involved in pre-mRNA splicing. In the fission yeast Schizosaccharomyces pombe, the U6 RNA gene was found to have an intron similar to a nuclear pre-mRNA intron, and it was proposed that the U6 intron might be inserted erroneously during pre-mRNA splicing. Using the polymerase chain reaction, we analyzed the U6 RNA genes of 52 organisms. In addition to the five species of Schizosaccharomyces, we found that the yeast species Rhodotorula hasegawae and Rhodosporidium dacryoidum also have mRNA-type introns in their U6 genes; however, in all the other organisms tested, we found no intron within the region of the U6 gene examined. Four introns and one intron are present in the R. hasegawae and R. dacryoidum U6 genes, respectively; and these introns are located at sites differing from the location of the Schizosaccharomyces U6 intron. Most of the U6 introns locate within the conserved domain, which is strikingly similar in structure to the catalytic center of the negative strand of the satellite RNA of tobacco ring spot virus. The introns of the S. pombe and R. dacryoidum U6 genes are located immediately adjacent to the nucleotides that were shown to be essential for the second step of the splicing reaction. These results support the notion that U6 RNA has a catalytic role in pre-mRNA splicing and that U6 introns originated from insertion of an excised intron during pre-mRNA splicing.     

Expression of CPEB,GAPDH and U6snRNA in cervical and ovarian tissue during cancer development

Persistent infection with high‐risk human papillomavirus (HPV) and expression of the proteins E6 and E7 is a prerequisite for development of cervical cancer. The distal non‐coding part of E6/E7 messengers from several HPV types is able to downregulate synthesis of a reporter gene through mechanisms with involvement of cytoplasmic polyadenylation elements (CPEs) in the messengers. We here show that the mRNA levels of one of the four known CPE‐binding proteins (CPEBs), the CPEB3, were downregulated in HPV‐positive cervical cancers, whereas in ovarian cancer the CPEB1 mRNA level was downregulated. In addition, we showed that the RNA levels of the widely used reference marker GAPDH were upregulated in both cancer forms, and the level of the reference marker U6snRNA was upregulated in cervical cancers. Moreover, a possible correlation between the degree of U6snRNA upregulation and cervical cancer propagation was shown. These changes observed in CPEB1 and CPEB3 might indicate regulatory functions of CPEBs in cancer development of HPV‐positive and HPV‐negative tumors, respectively, and the U6snRNA, GAPDH mRNA and CPEB1 mRNA levels may be useful as tumor markers for genital cancers although further investigations are needed.     

Immunoaffinity purification of a [U4/U6.U5] tri-snRNP from human cells.

We describe the isolation and biochemical characterization of [U4/U6.U5] tri-snRNP complexes from HeLa cells under nondenaturing conditions using a monoclonal antibody reacting with the U5-specific 100-kD protein. We show that the [U4/U6.U5] complex contains five previously unobserved proteins with molecular masses of 90, 60, 27, 20, and 15.5 kD, in addition to the core proteins, common to the U4/U6, U5, U1, and U2 snRNPs, and the U5-specific proteins, as found in 20S U5 snRNPs. With approximately 20 distinct snRNP proteins the complexity of the [U4/U6.U5] tri-snRNP is surprising. One or more of the five proteins found exclusively in the 25S [U4/U6.U5] tri-snRNP appears to be involved in the assembly of the tri-snRNP complex, as, in an in vitro reconstitution assay, purified 20S U5 and 10S U4/U6 snRNPs formed stable 25S [U4/U6.U5] complexes only in the presence of the free tri-snRNP-specific proteins. The formation of the [U4/U6.U5] complex in vitro does not require ATP, and the stability of the purified tri-snRNP complex is not affected by ATP to a measurable extent. However, the native [U4/U6.U5] displays a kinase activity that is absent in isolated U5: A 52-kD protein present in both U5 and [U4/U6.U5] is phosphorylated only in the latter. The function of this phosphorylation is unclear thus far; it may be involved in the activation of [U4/U6.U5] in the spliceosome.     

Alternative 3′-end processing of U5 snRNA by RNase III

The cellular components required to form the 3′ ends of small nuclear RNAs are unknown. U5 snRNA from Saccharomyces cerevisiae is found in two forms that differ in length at their 3′ ends (U5L and U5S). When added to a yeast cell free extract, synthetic pre-U5 RNA bearing downstream genomic sequences is processed efficiently and accurately to generate both mature forms of U5. The two forms of U5 are produced in vitro by alternative 3′-end processing. A temperature-sensitive mutation in the RNT1 gene encoding RNase III blocks accumulation of U5L in vivo. In vitro, alternative cleavage of the U5 precursor by RNase III determines the choice between the two multistep pathways that lead to U5L and U5S, one of which (U5L) is strictly dependent on RNase III. These results identify RNase III as a trans-acting factor involved in 3′-end formation of snRNA and show how RNase III might regulate alternative RNA processing pathways.