Albrecht Bindereif

A small-scale procedure for preparation of nuclear extracts that support efficient transcription and pre-mRNA splicing☆

A convenient and rapid method for preparing soluble extracts from the nuclei of as few as 3 x 10(7) mammalian cells (miniextract procedure) is described. By several criteria, miniextracts are comparable to nuclear extracts prepared from large numbers of cells by the conventional procedure. Miniextracts are able to support efficient transcription of a variety of class II promoters. In addition, DNase I footprinting and gel retardation assays can be performed directly in miniextracts, enabling the detection of sequence-specific DNA-binding proteins. Besides transcription, miniextracts efficiently carry out pre-mRNA splicing and allow formation and fractionation of previously characterized splicing complexes. The small-scale procedure enables simultaneous preparation of multiple extracts from a variety of cell types under different experimental conditions. Moreover, the use of small amounts of cells allows minimal expenditure of valuable or expensive materials such as radioactive compounds. Consequently, the procedure is highly advantageous for biochemical analysis of transcription and RNA processing in mammalian cells.

Exon Circularization Requires Canonical Splice Signals

Circular RNAs (circRNAs), an abundant class of noncoding RNAs in higher eukaryotes, are generated from pre-mRNAs by circularization of adjacent exons. Using a set of 15 circRNAs, we demonstrated their cell-type-specific expression and circular versus linear processing in mammalian cells. Northern blot analysis combined with RNase H cleavage conclusively proved a circular configuration for two examples, LPAR1 and HIPK3. To address the circularization mechanism, we analyzed the sequence requirements using minigenes derived from natural circRNAs. Both canonical splice sites are required for circularization, although they vary in flexibility and potential use of cryptic sites. Surprisingly, we found that no specific circRNA exon sequence is necessary and that potential flanking intron structures can modulate circularization efficiency. In combination with splice inhibitor assays, our results argue that the canonical spliceosomal machinery functions in circRNA biogenesis, constituting an alternative splicing mode.

Intronic CA-repeat and CA-rich elements: a new class of regulators of mammalian alternative splicing

We have recently identified an intronic polymorphic CA‐repeat region in the human endothelial nitric oxide synthase (eNOS) gene as an important determinant of the splicing efficiency, requiring specific binding of hnRNP L. Here, we analyzed the position requirements of this CA‐repeat element, which revealed its potential role in alternative splicing. In addition, we defined the RNA binding specificity of hnRNP L by SELEX: not only regular CA repeats are recognized with high affinity but also certain CA‐rich clusters. Therefore, we have systematically searched the human genome databases for CA‐repeat and CA‐rich elements associated with alternative 5′ splice sites (5′ss), followed by minigene transfection assays. Surprisingly, in several specific human genes that we tested, intronic CA RNA elements could function either as splicing enhancers or silencers, depending on their proximity to the alternative 5′ss. HnRNP L was detected specifically bound to these diverse CA elements. These data demonstrated that intronic CA sequences constitute novel and widespread regulatory elements of alternative splicing.

HnRNP L stimulates splicing of the eNOS gene by binding to variable-length CA repeats

In the human genome, dinucleotide repeats are common sequence elements of unknown functional significance. Here we demonstrate that CA repeats in intron 13 of the human endothelial nitric oxide synthase (eNOS) gene function as an unusual intronic splicing enhancer, whose activity depends on the CA repeat number. We identify the 65 kDa heterogenous nuclear ribonucleoprotein (hnRNP) L as the major factor that binds specifically and in a length-dependent manner to the CA-repeat enhancer. In addition, we show that hnRNP L functions as a specific activator of eNOS splicing, providing the first evidence for a role of hnRNP L in the regulation of mRNA splicing. We hypothesize that hnRNP L may be involved in the regulation of many other genes containing CA repeats or A/C-rich enhancers.

An ordered pathway of snRNP binding during mammalian pre-mRNA splicing complex assembly.

We have studied the assembly, composition and structure of splicing complexes using biotin‐avidin affinity chromatography and RNase protection assays. We find that U1, U2, U4, U5 and U6 snRNPs associate with the pre‐mRNA and are in the mature, functional complex. Association of U1 snRNP with the pre‐mRNA is rapid and ATP independent; binding of all other snRNPs occurs subsequently and is ATP dependent. Efficient binding of U1 and U2 snRNPs requires a 5′ splice site or a 3′ splice site/branch point region, respectively. Both sequence elements are required for efficient U4, U5 and U6 snRNP binding. Mutant RNA substrates containing only a 5′ splice site or a 3′ splice site/branch point region are assembled into ‘partial’ splicing complexes, which contain a subset of these five snRNPs. RNase protection experiments indicate that in contrast to U1 and U2 snRNPs, U4, U5 and U6 snRNPs do not contact the pre‐mRNA. Based upon the time course of snRNP binding and the composition of sucrose gradient fractionated splicing complexes we suggest an assembly pathway proceeding from a 20S (U1 snRNP only) through a 40S (U1 and U2 snRNPs) to the functional 60S splicing complex (U1, U2, U4, U5 and U6 snRNPs).

p110, a novel human U6 snRNP protein and U4/U6 snRNP recycling factor

During each spliceosome cycle, the U6 snRNA undergoes extensive structural rearrangements, alternating between singular, U4–U6 and U6–U2 base‐paired forms. In Saccharomyces cerevisiae, Prp24 functions as an snRNP recycling factor, reannealing U4 and U6 snRNAs. By database searching, we have identified a Prp24‐related human protein previously described as p110nrb or SART3. p110 contains in its C‐terminal region two RNA recognition motifs (RRMs). The N‐terminal two‐thirds of p110, for which there is no counterpart in the S.cerevisiae Prp24, carries seven tetratricopeptide repeat (TPR) domains. p110 homologs sharing the same domain structure also exist in several other eukaryotes. p110 is associated with the mammalian U6 and U4/U6 snRNPs, but not with U4/U5/U6 tri‐snRNPs nor with spliceosomes. Recombinant p110 binds in vitro specifically to human U6 snRNA, requiring an internal U6 region. Using an in vitro recycling assay, we demonstrate that p110 functions in the reassembly of the U4/U6 snRNP. In summary, p110 represents the human ortholog of Prp24, and associates only transiently with U6 and U4/U6 snRNPs during the recycling phase of the spliceosome cycle.

Auto- and Cross-Regulation of the hnRNP L Proteins by Alternative Splicing

ABSTRACT We recently characterized human hnRNP L as a global regulator of alternative splicing, binding to CA-repeat and CA-rich elements. Here we report that hnRNP L autoregulates its own expression on the level of alternative splicing. Intron 6 of the human hnRNP L gene contains a short exon that, if used, introduces a premature termination codon, resulting in nonsense-mediated decay (NMD). This “poison exon” is preceded by a highly conserved CA-rich cluster extending over 800 nucleotides that binds hnRNP L and functions as an unusually extended, intronic enhancer, promoting inclusion of the poison exon. As a result, excess hnRNP L activates NMD of its own mRNA, thereby creating a negative autoregulatory feedback loop and contributing to homeostasis of hnRNP L levels. We present experimental evidence for this mechanism, based on NMD inactivation, hnRNP L binding assays, and hnRNP L-dependent alternative splicing of heterologous constructs. In addition, we demonstrate that hnRNP L cross-regulates inclusion of an analogous poison exon in the hnRNP L-like pre-mRNA, which explains the reciprocal expression of the two closely related hnRNP L proteins.

HIGH CA REPEAT NUMBERS IN INTRON 13 OF THE ENDOTHELIAL NITRIC OXIDE SYNTHASE GENE AND INCREASED RISK OF CORONARY ARTERY DISEASE

Endothelial nitric oxide synthase (eNOS) plays a key role in vascular homeostasis. Because its product, nitric oxide, possesses vasodilatory and antiatherogenic properties, an altered eNOS function might promote atherosclerosis. We investigated the association between variations in CA repeat copy number [(CA), polymorphism] in intron 13 of the eNOS gene and the risk of coronary artery disease. (CA), polymorphism was investigated in 1000 consecutive patients with angiographically confirmed coronary artery disease and 1000 age- and gender-matched control subjects by a PCR-based fragment length calculation. Twenty-eight different alleles were identified containing 17-44 CA repeats. The presence of one allele containing > or = 38 repeats was associated with an excess risk of coronary artery disease (odds ratio 1.94, 95% confidence interval 1.31-2.86, P = 0.001). Carriers of alleles containing > or = 38 CA repeats were, in particular, overrepresented in the subgroup without common cardiovascular risk factors (odds ratio 3.39, 95% confidence interval 1.30-8.86, P = 0.009). Logistic regression analysis revealed that the (CA), polymorphism proved to be an independent risk factor (relative risk 2.17, 95% confidence interval 1.44-3.27, P = 0.0002). Our findings indicate that high numbers of CA repeats in intron 13 of the eNOS gene are associated with an excess risk of coronary artery disease.

Network of coregulated spliceosome components revealed by zebrafish mutant in recycling factor p110

The spliceosome cycle consists of assembly, catalysis, and recycling phases. Recycling of postspliceosomal U4 and U6 small nuclear ribonucleoproteins (snRNPs) requires p110/SART3, a general splicing factor. In this article, we report that the zebrafish earl grey (egy) mutation maps in the p110 gene and results in a phenotype characterized by thymus hypoplasia, other organ-specific defects, and death by 7 to 8 days postfertilization. U4/U6 snRNPs were disrupted in egy mutant embryos, demonstrating the importance of p110 for U4/U6 snRNP recycling in vivo. Surprisingly, expression profiling of the egy mutant revealed an extensive network of coordinately up-regulated components of the spliceosome cycle, providing a mechanism compensating for the recycling defect. Together, our data demonstrate that a mutation in a general splicing factor can lead to distinct defects in organ development and cause disease.

Trans mRNA splicing in trypanosomes: cloning and analysis of a PRP8‐homologous gene from Trypanosoma brucei provides evidence for a U5‐analogous RNP

In trypanosomes all mRNAs are generated through trans mRNA splicing, requiring the functions of the small nuclear RNAs U2, U4 and U6. In the absence of conventional cis mRNA splicing, the structure and function of a U5‐analogous snRNP in trypanosomes has remained an open question. In cis splicing, a U5 snRNP‐specific protein component called PRP8 in yeast and p220 in man is a highly conserved, essential splicing factor involved in splice‐site recognition and selection. We have cloned and sequenced a genomic region from Trypanosoma brucei, that contains a PRP8/p220‐homologous gene (p277) coding for a 277 kDa protein. Using an antibody against a C‐terminal region of the trypanosomal p277 protein, a small RNA of ∼65 nucleotides could be specifically co‐immunoprecipitated that appears to be identical with a U5 RNA (SLA2 RNA) recently identified by Dungan et al. (1996) . Based on sedimentation, immunoprecipitation and Western blot analyses we conclude that this RNA is part of a stable ribonucleoprotein (RNP) complex and associated not only with the p277 protein, but also with the common proteins present in the other trans‐spliceosomal snRNPs. Together these results demonstrate that a U5‐analogous RNP exists in trypanosomes and suggest that basic functions of the U5 snRNP are conserved between cis and trans splicing.