哺乳动物细胞mRNA剪接调控的分子机制

mRNA的可变剪接是指一个单一的mRNA前体(pre-mRNA)经过不同的剪接加工方式生成多种mRNA变异体(variants)的过程,这些变异体最终可以编码合成具有不同结构和功能的蛋白质。在过去的10多年中,大量数据表明,可变剪接是增加转录组和蛋白质组多样性的重要资源,也是调控哺乳动物细胞基因表达的重要步骤。可变剪接具有高度的组织与发育阶段特异性,并受到外界信号的控制。剪接调控的紊乱与疾病的发生发展密切相关。该文将对哺乳动物细胞mRNA剪接调控的分子机制进行阐述。     

In vivo cytogenetics: mammalian germ cells

This chapter summarizes the most relevant methodologies available for evaluation of cytogenetic damage induced in vivo in mammalian germ cells. Protocols are provided for the following endpoints: numerical and structural chromosome aberrations in secondary oocytes or first-cleavage zygotes, reciprocal translocations in primary spermatocytes, chromosome counting in secondary spermatocytes, numerical and structural chromosome aberrations, and sister chromatid exchanges (SCE) in spermatogonia, micronuclei in early spermatids, aneuploidy in mature sperm. The significance of each methodology is discussed. The contribution of novel molecular cytogenetic approaches to the detection of chromosome damage in rodent germ cells is also considered.     

Analysis of the stimulatory effect of splicing on mRNA production and utilization in mammalian cells

We have examined how splicing affects the expression of a range of human and nonhuman genes in vertebrate cells. Although our data demonstrate that splicing invariably enhances the level of gene expression, this positive effect is generally moderate. However, in the case of the human beta-globin gene, splicing is essential for significant protein expression. In the absence of introns, 3' end processing is inefficient, and this appears to be causally linked to a significant decrease in the level of both nuclear and cytoplasmic 3' end-processed RNA. In contrast, splicing appears to only modestly enhance nuclear mRNA export. Consistent with this observation, intronless beta-globin gene expression was only partially rescued by the insertion of retroviral nuclear mRNA export elements. Surprisingly, in the case of the highly intron dependent beta-globin gene, the mRNA that did reach the cytoplasm was also only inefficiently translated if it derived from an intronless expression plasmid. Together, these data argue that splicing can increase gene expression by enhancing mRNA 3' end processing, and hence, mRNA production. Moreover, in the case of the highly intron-dependent beta-globin gene, splicing also significantly enhanced the translational utilization of cytoplasmic beta-globin mRNAs.     

In vivo cross-linking of proteins to mRNA in human cells

Human KB cells were irradiated with ultraviolet light to cross-link mRNA to its associated proteins. More than 75% of both the poly(A)-containing and the poly(A)-lacking mRNAs were cross-linked to proteins after 3 min irradiation. Glycerol gradient analysis showed that no significant RNA chain breakage occurred during this treatment. Cross-linked poly(A)-containing mRNA-protein complexes were purified by oligo(dT)cellulose chromatography in the presence of sodium dodecylsulphate. CsCl gradient analysis revealed that the low salt eluted particles had a buoyant density of about 1.47 g/cm³. To determine which proteins were cross-linked to mRNA, covalent mRNA-protein complexes, labeled in their RNA moiety, were exhaustively treated with nucleases. Polyacrylamide gel analysis showed that most of the residual RNA-radioactivity was covalently bound to proteins of 73000, 69000 and 52000 molecular weight.     

A double-reporter splicing assay for determining splicing efficiency in mammalian cells

Changes in alternative splicing patterns can result from both inherited and acquired defects, and they are increasingly recognized as causes of human diseases. Hence, improvements in the understanding of alternative splicing regulation may provide opportunities for restoring productive patterns of splicing. The identification of factors (such as proteins, nucleic acids or small molecules) that modulate the splicing pattern would be facilitated by systems with which many samples can be screened. The absence of reliable systems prompted us to develop an assay system based on dual enzymatic activities. Two distinct signals derived from spliced and unspliced RNA are measured, providing the basis for a robust, rapid and convenient assay for investigating splicing. This protocol describes how to use this system; the time required for lysing the cells and recording enzymatic activity is about 2 hours.     

In vivo imaging of oskar mRNA transport reveals the mechanism of posterior localization

oskar mRNA localization to the posterior of the Drosophila oocyte defines where the abdomen and germ cells form in the embryo. Although this localization requires microtubules and the plus end-directed motor, kinesin, its mechanism is controversial and has been proposed to involve active transport to the posterior, diffusion and trapping, or exclusion from the anterior and lateral cortex. By following oskar mRNA particles in living oocytes, we show that the mRNA is actively transported along microtubules in all directions, with a slight bias toward the posterior. This bias is sufficient to localize the mRNA and is reversed in mago, barentsz, and Tropomyosin II mutants, which mislocalize the mRNA anteriorly. Since almost all transport is mediated by kinesin, oskar mRNA localizes by a biased random walk along a weakly polarized cytoskeleton. We also show that each component of the oskar mRNA complex plays a distinct role in particle formation and transport.     

Citrate transport and metabolism in mammalian cells

Citrate, an organic trivalent anion, is a major substrate for generation of energy in most cells. It is produced in mitochondria and used either in the Krebs' cycle or released into cytoplasm through a specific mitochondrial carriers. Citrate can also be taken up from blood through different plasma membrane transporters. In the cytoplasm, citrate can be used ultimately for fatty acid synthesis, which is increased in cancer cells. Here, we review the ways in which citrate can be transported and discuss the changes in transport and metabolism that occur in cancer cells. The primary focus is on the prostate gland, which is known to produce and release large amounts of citrate during its normal secretory function. The significant changes that occur in citrate‐related metabolism and transport in prostate cancer are the second focus. This review strives to relate these mechanisms to molecular biology on the one hand and to clinical applications on the other.     

In vivo detection of snRNP-rich organelles in the nuclei of mammalian cells.

The in vivo distribution of snRNPs has been analysed by microinjecting fluorochrome-labelled antisense probes into the nuclei of live HeLa and 3T3 cells. Probes for U2 and U5 snRNAs specifically label the same discrete nuclear foci while a probe for U1 snRNA shows widespread nucleoplasmic labelling, excluding nucleoli, in addition to labelling foci. A probe for U3 snRNA specifically labels nucleoli. These in vivo data confirm that mammalian cells have nuclear foci which contain spliceosomal snRNPs. Co-localization studies, both in vivo and in situ, demonstrate that the spliceosomal snRNAs are present in the same nuclear foci. These foci are also stained by antibodies which recognize snRNP proteins, m3G-cap structures and the splicing factor U2AF but are not stained by anti-SC-35 or anti-La antibodies. U1 snRNP and the splicing factor U2AF closely co-localize in the nucleus, both before and after actinomycin D treatment, suggesting that they may both be part of the same complex in vivo.     

Regulation of mRNA stability in mammalian cells

The regulation of mRNA decay is a major control point in gene expression. The stability of a particular mRNA is controlled by specific interactions between its structural elements and RNA-binding proteins that can be general or mRNA-specific. Regulated mRNA stability is achieved through fluctuations in half-lives in response to developmental or environmental stimuli like nutrient levels, cytokines, hormones and temperature shifts as well as environmental stresses like hypoxia, hypocalcemia, viral infection, and tissue injury. Furthermore, in specific disorders like some forms of neoplasia, thalassemia and Alzheimer's disease, deregulated mRNA stability can lead to the aberrant accumulation of mRNAs and the proteins they encode. This review presents a discussion of some recently identified examples of regulated and deregulated mRNA stability in order to illustrate the diversity of genes regulated by alterations in the degradation rates of their mRNAs.