Structural architecture of the human long non-coding RNA, steroid receptor RNA activator

While functional roles of several long non-coding RNAs (lncRNAs) have been determined, the molecular mechanisms are not well understood. Here, we report the first experimentally derived secondary structure of a human lncRNA, the steroid receptor RNA activator (SRA), 0.87 kB in size. The SRA RNA is a non-coding RNA that coactivates several human sex hormone receptors and is strongly associated with breast cancer. Coding isoforms of SRA are also expressed to produce proteins, making the SRA gene a unique bifunctional system. Our experimental findings (SHAPE, in-line, DMS and RNase V1 probing) reveal that this lncRNA has a complex structural organization, consisting of four domains, with a variety of secondary structure elements. We examine the coevolution of the SRA gene at the RNA structure and protein structure levels using comparative sequence analysis across vertebrates. Rapid evolutionary stabilization of RNA structure, combined with frame-disrupting mutations in conserved regions, suggests that evolutionary pressure preserves the RNA structural core rather than its translational product. We perform similar experiments on alternatively spliced SRA isoforms to assess their structural features.     

Alternative intronic promoters in development and disease

Approximately 20,000 mammalian genes are estimated to encode between 250 thousand and 1 million different proteins. This enormous diversity of the mammalian proteome is caused by the ability of a single-gene locus to encode multiple protein isoforms. Protein isoforms encoded by one gene locus can be functionally distinct, and they can even have antagonistic functions. One of the mechanisms involved in creating this proteome complexity is alternative promoter usage. Alternative intronic promoters are located downstream from their canonical counterparts and drive the expression of alternative RNA isoforms that lack upstream exons. These upstream exons can encode some important functional domains, and proteins encoded by alternative mRNA isoforms can be thus functionally distinct from the full-length protein encoded by canonical mRNA isoforms. Since any misbalance of functionally distinct protein isoforms is likely to have detrimental consequences for the cell and the whole organism, their expression must be precisely regulated. Misregulation of alternative intronic promoters is frequently associated with various developmental defects and diseases including cancer, and it is becoming increasingly clear that this phenomenon deserves more attention.     

Two isoforms of the T-cell intracellular antigen 1 (TIA-1) splicing factor display distinct splicing regulation activities. Control of TIA-1 isoform ratio by TIA-1-related protein

TIA-1 (T-cell Intracellular Antigen 1) and TIAR (TIA-1-related protein) are RNA-binding proteins involved in the regulation of alternative pre-mRNA splicing and other aspects of RNA metabolism. Various isoforms of these proteins exist in mammals. For example, TIA-1 presents two major isoforms (TIA-1a and TIA-1b) generated by alternative splicing of exon 5 that differ by eleven amino acids exclusive of the TIA-1a isoform. Here we show that the relative expression of TIA-1 and TIAR isoforms varies in different human tissues and cell lines, suggesting distinct functional properties and regulated isoform expression. We report that whereas TIA-1 isoforms show similar subcellular distribution and RNA binding, TIA-1b displays enhanced splicing stimulatory activity compared with TIA-1a, both in vitro and in vivo. Interestingly, TIAR depletion from HeLa and mouse embryonic fibroblasts results in an increased ratio of TIA-1b/a expression, suggesting that TIAR regulates the relative expression of TIA-1 isoforms. Taken together, the results reveal distinct functional properties of TIA-1 isoforms and the existence of a regulatory network that controls isoform expression.     

SRA的RNA和蛋白质均有转录辅激活功能

类固醇受体激活物（steroid receptor activator, SRA）是一种 类固醇受体辅激活物.最初的研究认为,SRA只存在RNA形式,不存在蛋白质 形式.但是后来的研究发现,SRA是在RNA和蛋白质两个水平上发挥功能的分 子,其cDNA序列存在687 bp保守的核心区域,该核心区域对其发挥转录共激 活活性是必需的.SRA的RNA形式主要参与核受体的转录共激活作用,其表达 与乳腺癌的发生有很大关系,SRA的蛋白质形式（steroid receptor activator protein ,SRAP）也具有类似的功能.但是不同于RNA形式, SRAP可结合到特定基因的启动子区域,并起到阻遏物的作用.本文对SRA的 特点、表达及功能等方面的最新研究进展及其可能的作用机制与作用形式 进行概述.     

Alternatively spliced protein arginine methyltransferase 1 isoform PRMT1v2 promotes the survival and invasiveness of breast cancer cells

Protein arginine methylation is catalyzed by protein arginine methyltransferases (PRMTs) and plays an important role in many cellular processes. Aberrant PRMT expression has been observed in several common cancer types; however, their precise contribution to the cell transformation process is not well understood. We previously reported that the PRMT1 gene generates several alternatively spliced isoforms, and our initial biochemical characterization of these isoforms revealed that they exhibit distinct substrate specificity and subcellular localization. We focus here on the PRMT1v2 isoform, which is the only predominantly cytoplasmic isoform, and we have found that its relative expression is increased in breast cancer cell lines and tumors. Specific depletion of PRMT1v2 using RNA interference caused a significant decrease in cancer cell survival due to an induction of apoptosis. Furthermore, depletion of PRMT1v2 in an aggressive cancer cell line significantly decreased cell invasion. We also demonstrate that PRMT1v2 overexpression in a non-aggressive cancer cell line was sufficient to render them more invasive. Importantly, this novel activity is specific to PRMT1v2, as overexpression of other isoforms did not enhance invasion. Moreover, this activity requires both proper subcellular localization and methylase activity. Lastly, PRMT1v2 overexpression altered cell morphology and reduced cell-cell adhesion, a phenomenon that we convincingly linked with reduced β-catenin protein expression. Overall, we demonstrate a specific role for PRMT1v2 in breast cancer cell survival and invasion, underscoring the importance of identifying and characterizing the distinct functional differences between PRMT1 isoforms.     

Cluster analysis of an extensive human breast cancer cell line protein expression map database

In the current study, the protein expression maps (PEMs) of 26 breast cancer cell lines and three cell lines derived from normal breast or benign disease tissue were visualised by high resolution two-dimensional gel electrophoresis. Analysis of this data was performed with ChiClust and ChiMap, two analytical bioinformatics tools that are described here. These tools are designed to facilitate recognition of specific patterns shared by two or more (a series) PEMs. Both tools use PEMs that were matched by an image analysis program and locally written programs to create a match table that is saved in an object relational database. The ChiClust tool uses clustering and subclustering methods to extract statistically significant protein expression patterns from a large series of PEMs. The ChiMap tool calculates a differential value (either as percentage change or a fold change) and represents these graphically. All such differentials or just those identified using ChiClust can be submitted to ChiMap. These methods are not dependent on any particular commercial image analysis program, and the whole software package gives an integrated procedure for the comparison and analysis of a series of PEMs. The ChiClust tool was used here to order the breast cell lines into groups according to biological characteristics including morphology in vitro and tumour forming ability in vivo. ChiMap was then used to highlight eight major protein feature-changes detected between breast cancer cell lines that either do or do not proliferate in nude mice. Mass spectrometry was used to identify the proteins. The possible role of these proteins in cancer is discussed.     

The effect of a DNA repair gene on cellular invasiveness: XRCC3 over-expression in breast cancer cells

Over-expression of DNA repair genes has been associated with resistance to radiation and DNA-damage induced by chemotherapeutic agents such as cisplatin. More recently, based on the analysis of genome expression profiling, it was proposed that over-expression of DNA repair genes enhances the invasive behaviour of tumour cells. In this study we present experimental evidence utilizing functional assays to test this hypothesis. We assessed the effect of the DNA repair proteins known as X-ray complementing protein 3 (XRCC3) and RAD51, to the invasive behavior of the MCF-7 luminal epithelial-like and BT20 basal-like triple negative human breast cancer cell lines. We report that stable or transient over-expression of XRCC3 but not RAD51 increased invasiveness in both cell lines in vitro. Moreover, XRCC3 over-expressing MCF-7 cells also showed a higher tumorigenesis in vivo and this phenotype was associated with increased activity of the metalloproteinase MMP-9 and the expression of known modulators of cell-cell adhesion and metastasis such as CD44, ID-1, DDR1 and TFF1. Our results suggest that in addition to its' role in facilitating repair of DNA damage, XRCC3 affects invasiveness of breast cancer cell lines and the expression of genes associated with cell adhesion and invasion.     

Comprehensive analysis of cellular galectin-3 reveals no consistent oncogenic function in pancreatic cancer cells

Galectin-3 (Gal-3), a 31 kDa member of the family of beta-galactoside-binding proteins, has been implicated in the progression of different human cancers. However, the proposed roles differ widely, ranging from tumor-promoting cellular functions and negative impact on patient prognosis to tumor-suppressive properties and positive prognostic impact. We and others have previously identified Gal-3 as overexpressed in pancreatic cancer as compared to chronic pancreatitis and normal pancreatic tissue. The purpose of this study was thus the comprehensive analysis of putative cellular functions of Gal-3 by transient as well as stable silencing or overexpression of Gal-3 in a panel of 6 well-established pancreatic cancer cell lines. Our results confirm that galectin-3 is upregulated at the mRNA level in pancreatic cancer and strongly expressed in the majority of pancreatic cancer cell lines. In individual cell lines, transient knockdown of Gal-3 expression resulted in moderate inhibitory effects on proliferation, migration or anchorage-independent growth of the cells, but these effects were not consistent across the spectrum of analyzed cell lines. Moreover, functional effects of the modulation of Gal-3 expression were not observed in stable knockdown or overexpression approaches in vitro and did not alter the growth characteristics of nude mouse xenograft tumors in vivo. Our data thus do not support a direct functional role of Gal-3 in the malignant transformation of pancreatic epithelial cells, although paracrine or systemic effects of Gal-3 expression are not excluded.     

A secreted glycoprotein induced by estrogen in human breast cancer cell lines

Estrogen induces the synthesis of a glycoprotein of molecular weight 46,000 daltons in three estrogen receptor-positive breast cancer cell lines (MCF₇, ZR₇₅ and T47D), but not in an estrogen receptor-negative cell line (BT 20) or a nonmalignant cell line (HBL 100). The 46K protein, which accounts for 40% of ³⁵S-methionine incorporation into secreted proteins, is only induced by steroids able to interact with the estrogen receptor. The anti-estrogens tamoxifen and hydroxytamoxifen, which by themselves were inactive, suppressed the induction of this protein by estradiol. In MCF₇ cells, estradiol also induces three intracellular proteins which are resolved in two-dimensional electrophoresis. The induction of the 46K secreted protein(s) makes these cell lines excellent in vitro systems for studying the mechanism of estrogen and anti-estrogen action. This protein may also be a useful probe for studying the action of estrogen on breast cancer growth, and may be a useful marker for predicting the hormonal responsiveness of breast cancer in vivo.