染色质结构与mRNA可变剪接

mRNA的可变剪接(alternative splicing)是一种由一个mRNA前体(pre-mRNA)通过不同的剪接方式产生多个mRNA变异体(variants)的RNA加工过程。在过去很长一段时间里,人们认为mRNA剪接过程是独立于转录过程的一个转录后RNA加工过程。然而,越来越多的实验证明mRNA剪接在很大程度上是与转录偶联发生的。因此,剪接调控会受到与转录相关因素的调控。本文将对染色质与mRNA剪接调控的相关性和染色质结构调控可变剪接的分子机制进行阐述。     

Associations between distinct pre-mRNA splicing components and the cell nucleus.

SC-35 is a non-snRNP spliceosome component that is specifically recognized by the anti-spliceosome monoclonal antibody alpha SC-35. In this paper we provide direct evidence that SC-35 is an essential splicing factor and we examine the immunolocalization of SC-35 by confocal laser scanning microscopy and by electron microscopy. We have found that the speckled staining pattern observed by fluorescence microscopy corresponds to structures previously designated as interchromatin granules and perichromatin fibrils. Although snRNP antigens are also concentrated in these nuclear regions, we show that the two types of spliceosome components are localized through different molecular interactions: The distribution of SC-35 was not affected by treatment with DNase I or RNase A, or when the cells were heat shocked. In contrast, snRNP antigens become diffusely distributed after RNase A digestion or heat shock. Examination of cells at different stages of mitosis revealed that the SC-35 speckled staining pattern is lost during prophase and speckles containing SC-35 begin to reform in the cytoplasm of anaphase cells. In contrast, snRNP antigens do not associate with speckled regions until late in telophase. These studies reveal a dynamic pattern of assembly and disassembly of the splicing factor SC-35 into discrete nuclear structures that colocalize with interchromatin granules and perichromatin fibrils. These subnuclear regions may therefore be nuclear organelles involved in the assembly of spliceosomes, or splicing itself.     

Nucleotide substitutions within the cardiac troponin T alternative exon disrupt pre-mRNA alternative splicing.

The cardiac troponin T (cTNT) pre-mRNA contains a single alternative exon (exon 5) which is either included or excluded from the processed mRNA. Using transient transfection of cTNT minigenes, we have previously localized pre-mRNA cis elements required for exon 5 alternative splicing to three small regions of the pre-mRNA which include exons 4, 5, and 6. In the present study, nucleotide substitutions were introduced into the region containing exon 5 to begin to define specific nucleotides required for exon 5 alternative splicing. A mutation within the 5' splice site flanking the cTNT alternative exon that increases its homology to the consensus sequence improves splicing efficiency and leads to increased levels of mRNAs that include the alternative exon. Surprisingly, substitution of as few as four nucleotides within the alternative exon disrupts cTNT pre-mRNA alternative splicing and prevents recognition of exon 5 as a bona fide exon. These results establish that the cTNT alternative exon contains information in cis that is required for its recognition by the splicing machinery.     

Interactions between introns via exon definition in plant pre-mRNA splicing

The barley gene Mlo encodes a prototype of a novel class of plant proteins. In mlo mutants, absence of the 60 kDa wild-type Mlo protein results in broad-spectrum resistance to the powdery mildew fungus, Erysiphe graminis f. sp. hordei . To directly assess its function, Mlo was transiently expressed with a marker gene encoding a modified green fluorescent protein (GFP) in leaf epidermal cells of mlo resistant barley lines. Fungal inoculation of epidermal cells transfected with wild-type Mlo led to haustorium formation and abundant sporulation. Therefore, expression of the wild-type Mlo gene, in mlo resistant genotypes, is both necessary and sufficient to restore susceptibility to fungal attack. Complementation of mlo resistance alleles was restricted to single host cells, indicating a cell-autonomous function for the wild-type Mlo protein. We discuss our findings with respect to source–sink relationships of plants and biotrophic fungi and the potentially wide-ranging use of the transient complementation assay to analyse host compatibility and defence in response to powdery mildew attack.     

Human pre-mRNA splicing signals.

A sample of 764 pairs of human pre-mRNA exon-intron and intron-exon boundaries, extracted from the European Molecular Biology Laboratory data bank, is analyzed to provide a species-optimized characterization of donor and acceptor sites, evaluate the information content of the two signals (found to be about 8 and 9 bits respectively) and check the independent-base approximation (which holds well) and the "GT-AG" rule (to which, a few well-documented exceptions are found). No correlation is detected between the strength ("discrimination energy") of an actual donor-site signal and that of its corresponding acceptor-site counterpart, nor between that of either signal, or the cumulative strength of both, and the length of the intervening intron. The discrimination-energy distributions of the two signals are determined. Because of the large sample size and its single-species origin, the two distributions can be presumed to be representative of their underlying genomic counterparts. The size distribution of the introns shows a lower cut-off of 70 nucleotides (in essential agreement with published experimental results), and apparently no periodicities. A smaller sample of mammalian branch sites, taken from the literature, is similarly analyzed to attempt a characterization of this rather elusive signal, and provides some indication that at least part of the "long pyrimidine stretch", usually considered an integral constituent of the 3' splice signal, may be just as strongly associated with the branch site, in agreement with recent experimental observations. The usefulness of these characterizations for splice-junction searches is assessed on a test sequence.     

Temperature-dependent splicing of beta-globin pre-mRNA

A T→G mutation at nucleotide 705 of human β-globin intron 2 creates an aberrant 5′ splice site and activates a cryptic 3′ splice site upstream. In consequence, the pre-mRNA is spliced via aberrant splice sites, despite the presence of the still functional correct sites. Surprisingly, when IVS2-705 HeLa or K562 cells were cultured at temperatures below 30°C, aberrant splicing was inhibited and correct splicing was restored. Similar temperature effects were seen for another β-globin pre-mRNA, IVS2-745, and in a construct in which a β-globin intron was inserted into a coding sequence of EGFP. Temperature-induced alternative splicing was affected by the nature of the internal aberrant splice sites flanking the correct sites and by exonic sequences. The results indicate that in the context of thalassemic splicing mutations and possibly in other alternatively spliced pre-mRNAs, temperature is one of the parameters that affect splice site selection.     

Epigenetics in alternative pre-mRNA splicing

Alternative splicing plays critical roles in differentiation, development, and disease and is a major source for protein diversity in higher eukaryotes. Analysis of alternative splicing regulation has traditionally focused on RNA sequence elements and their associated splicing factors, but recent provocative studies point to a key function of chromatin structure and histone modifications in alternative splicing regulation. These insights suggest that epigenetic regulation determines not only what parts of the genome are expressed but also how they are spliced.     

snRNAs as the catalysts of pre-mRNA splicing

The spliceosome, the gigantic molecular machine that performs pre-mRNA splicing in eukaryotes, contains over 200 different proteins and five RNA molecules. The central role played by the spliceosomal RNAs in splicing has led to the hypothesis that, like the ribosome, the spliceosome is an RNA-centric enzyme and a relic from the RNA world. Recent structural studies have provided the first glimpses of the structural features of spliceosomal RNAs, and mutational analyses in vivo and in vitro have uncovered new functional roles for a catalytically essential domain. An emerging model for the active site of group II introns, a closely related class of natural ribozymes, is likely to provide a wealth of insights on structure and function of the active site of the spliceosome.     

Interplay between proliferation and differentiation within the myogenic lineage.

In muscle cells, as in a variety of cell types, proliferation and differentiation are mutually exclusive events controlled by a balance of opposing cellular signals. Members of the MyoD family of muscle-specific helix-loop-helix proteins which, in collaboration with ubiquitous factors, activate muscle differentiation and inhibit cell proliferation function at the nexus of the cellular circuits that control proliferation and differentiation of muscle cells. The activities of these myogenic regulators are negatively regulated by peptide growth factors and activated oncogenes whose products transmit growth signals from the membrane to the nucleus. Recent studies have revealed multiple mechanisms through which intracellular growth factor signals may interfere with the functions of the myogenic regulators. When expressed at high levels, members of the MyoD family can override mitogenic signals and can cause growth arrest independent of their effects on differentiation. The ability of these myogenic regulators to inhibit proliferation of normal as well as transformed cells from multiple lineages suggests that they interact with conserved components of the cellular machinery involved in cell cycle progression and that similar types of regulatory factors participate in differentiation and cell cycle control in diverse cell types.     

Interplay between AAUAAA and the trans-splice site in processing of a Caenorhabditis elegans operon pre-mRNA

About half of Caenorhabditis elegans genes have a 1-2 bp mismatch to the canonical AAUAAA hexamer that signals 3' end formation. One rare variant, AGUAAA, is found at the 3' end of the mai-1 gene, the first gene in an operon also containing gpd-2 and gpd-3. When we expressed this operon under heat shock control, 3' end formation dependent on the AGUAAA was very inefficient, but could be rescued by a single bp change to create a perfect AAUAAA. When AGUAAA was present, most 3' ends formed at a different site, 100 bp farther downstream, right at the gpd-2 trans-splice site. Surprisingly, 3' end formation at this site did not require any observable match to the AAUAAA consensus. It is possible that 3' end formation at this site occurs by a novel mechanism--trans-splicing-dependent cleavage--as deletion of the trans-splice site prevented 3' end formation here. Changing the AGUAAA to AAUAAA also influenced the trans-splicing process: with AGUAAA, most of the gpd-2 product was trans-spliced to SL1, rather than SL2, which is normally used at downstream operon trans-splice sites. However, with AAUAAA, SL2 trans-splicing of gpd-2 was increased. Our results imply that (1) the AAUAAA consensus controls 3' end formation frequency in C. elegans; (2) the AAUAAA is important in determining SL2 trans-splicing events more than 100 bp downstream; and (3) in some circumstances, 3' end formation may occur by a trans-splicing-dependent mechanism.     

Structural aspects of microRNA biogenesis

One of the biggest surprises at the beginning of the 'post-genome era' was the discovery of numerous genes encoding microRNAs. They were found in genomes of such diverse organisms as Caenorhabditis elegans, Drosophila melanogaster, Arabidopsis thaliana, and Homo sapiens which implies their important role in multicellular life evolution. The number of microRNA genes is estimated to be nearly 1% of that of protein-coding genes. Their products, tiny RNAs, are thought to regulate gene expression during development, organogenesis, and very likely during many other processes, by hybridizing to their target mRNAs. The cellular functions of mRNAs that are regulated by microRNAs are only beginning to be revealed, and details of this regulation mechanism are still poorly understood. In this article we discuss the possible mechanisms of microRNA biogenesis with special emphasis on their structural aspects. We have focused on the factors and effects that may be responsible for the existing length differences between different microRNAs, and for the observed length heterogeneity within some individual microRNA species.