Deciphering human ribonucleoprotein regulatory networks

RNA-binding proteins (RBPs) control and coordinate each stage in the life cycle of RNAs. Although in vivo binding sites of RBPs can now be determined genome-wide, most studies typically focused on individual RBPs. Here, we examined a large compendium of 114 high-quality transcriptome-wide in vivo RBP-RNA cross-linking interaction datasets generated by the same protocol in the same cell line and representing 64 distinct RBPs. Comparative analysis of categories of target RNA binding preference, sequence preference, and transcript region specificity was performed, and identified potential posttranscriptional regulatory modules, i.e. specific combinations of RBPs that bind to specific sets of RNAs and targeted regions. These regulatory modules represented functionally related proteins and exhibited distinct differences in RNA metabolism, expression variance, as well as subcellular localization. This integrative investigation of experimental RBP-RNA interaction evidence and RBP regulatory function in a human cell line will be a valuable resource for understanding the complexity of post-transcriptional regulation

An Introductory Guide to Aligning Networks Using SANA, the Simulated Annealing Network Aligner.

Sequence alignment has had an enormous impact on our understanding of biology, evolution, and disease. The alignment of biological networks holds similar promise. Biological networks generally model interactions between biomolecules such as proteins, genes, metabolites, or mRNAs. There is strong evidence that the network topology-the "structure" of the network-is correlated with the functions performed, so that network topology can be used to help predict or understand function. However, unlike sequence comparison and alignment-which is an essentially solved problem-network comparison and alignment is an NP-complete problem for which heuristic algorithms must be used.Here we introduce SANA, the Simulated Annealing Network Aligner. SANA is one of many algorithms proposed for the arena of biological network alignment. In the context of global network alignment, SANA stands out for its speed, memory efficiency, ease-of-use, and flexibility in the arena of producing alignments between two or more networks. SANA produces better alignments in minutes on a laptop than most other algorithms can produce in hours or days of CPU time on large server-class machines. We walk the user through how to use SANA for several types of biomolecular networks

Identification of the RNA recognition element of the RBPMS family of RNA-binding proteins and their transcriptome-wide mRNA targets

Recent studies implicated the RNA-binding protein with multiple splicing (RBPMS) family of proteins in oocyte, retinal ganglion cell, heart, and gastrointestinal smooth muscle development. These RNA-binding proteins contain a single RNA recognition motif (RRM), and their targets and molecular function have not yet been identified. We defined transcriptome-wide RNA targets using photoactivatable-ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) in HEK293 cells, revealing exonic mature and intronic pre-mRNA binding sites, in agreement with the nuclear and cytoplasmic localization of the proteins. Computational and biochemical approaches defined the RNA recognition element (RRE) as a tandem CAC trinucleotide motif separated by a variable spacer region. Similar to other mRNA-binding proteins, RBPMS family of proteins relocalized to cytoplasmic stress granules under oxidative stress conditions suggestive of a support function for mRNA localization in large and/or multinucleated cells where it is preferentially expressed

Evaluation of two commercial global miRNA expression profiling platforms for detection of less abundant miRNAs

<p>Abstract</p> <p>Background</p> <p>microRNAs (miRNA) are short, endogenous transcripts that negatively regulate the expression of specific mRNA targets. miRNAs are found both in tissues and body fluids such as plasma. A major perspective for the use of miRNAs in the clinical setting is as diagnostic plasma markers for neoplasia. While miRNAs are abundant in tissues, they are often scarce in plasma. For quantification of miRNA in plasma it is therefore of importance to use a platform with high sensitivity and linear performance in the low concentration range. This motivated us to evaluate the performance of three commonly used commercial miRNA quantification platforms: GeneChip miRNA 2.0 Array, miRCURY Ready-to-Use PCR, Human panel I+II V1.M, and TaqMan Human MicroRNA Array v3.0.</p> <p>Results</p> <p>Using synthetic miRNA samples and plasma RNA samples spiked with different ratios of 174 synthetic miRNAs we assessed the performance characteristics reproducibility, recovery, specificity, sensitivity and linearity. It was found that while the qRT-PCR based platforms were sufficiently sensitive to reproducibly detect miRNAs at the abundance levels found in human plasma, the array based platform was not. At high miRNA levels both qRT-PCR based platforms performed well in terms of specificity, reproducibility and recovery. At low miRNA levels, as in plasma, the miRCURY platform showed better sensitivity and linearity than the TaqMan platform.</p> <p>Conclusion</p> <p>For profiling clinical samples with low miRNA abundance, such as plasma samples, the miRCURY platform with its better sensitivity and linearity would probably be superior.</p

A Membrane-Bound Vertebrate Globin

The family of vertebrate globins includes hemoglobin, myoglobin, and other O2-binding proteins of yet unclear functions. Among these, globin X is restricted to fish and amphibians. Zebrafish (Danio rerio) globin X is expressed at low levels in neurons of the central nervous system and appears to be associated with the sensory system. The protein harbors a unique N-terminal extension with putative N-myristoylation and S-palmitoylation sites, suggesting membrane-association. Intracellular localization and transport of globin X was studied in 3T3 cells employing green fluorescence protein fusion constructs. Both myristoylation and palmitoylation sites are required for correct targeting and membrane localization of globin X. To the best of our knowledge, this is the first time that a vertebrate globin has been identified as component of the cell membrane. Globin X has a hexacoordinate binding scheme and displays cooperative O2 binding with a variable affinity (P50∼1.3–12.5 torr), depending on buffer conditions. A respiratory function of globin X is unlikely, but analogous to some prokaryotic membrane-globins it may either protect the lipids in cell membrane from oxidation or may act as a redox-sensing or signaling protein

Modulations of cell cycle checkpoints during HCV associated disease

Background Impaired proliferation of hepatocytes has been reported in chronic Hepatitis C virus infection. Considering the fundamental role played by cell cycle proteins in controlling cell proliferation, altered regulation of these proteins could significantly contribute to HCV disease progression and subsequent hepatocellular carcinoma (HCC). This study aimed to identify the alterations in cell cycle genes expression with respect to early and advanced disease of chronic HCV infection. Methods Using freshly frozen liver biopsies, mRNA levels of 84 cell cycle genes in pooled RNA samples from patients with early or advanced fibrosis of chronic HCV infection were studied. To associate mRNA levels with respective protein levels, four genes (p27, p15, KNTC1 and MAD2L1) with significant changes in mRNA levels (\u3e 2-fold, p-value \u3c 0.05) were selected, and their protein expressions were examined in the liver biopsies of 38 chronic hepatitis C patients. Results In the early fibrosis group, increased mRNA levels of cell proliferation genes as well as cell cycle inhibitor genes were observed. In the advanced fibrosis group, DNA damage response genes were up-regulated while those associated with chromosomal stability were down-regulated. Increased expression of CDK inhibitor protein p27 was consistent with its mRNA level detected in early group while the same was found to be negatively associated with liver fibrosis. CDK inhibitor protein p15 was highly expressed in both early and advanced group, but showed no correlation with fibrosis. Among the mitotic checkpoint regulators, expression of KNTC1 was significantly reduced in advanced group while MAD2L1 showed a non-significant decrease. Conclusion Collectively these results are suggestive of a disrupted cell cycle regulation in HCV-infected liver. The information presented here highlights the potential of identified proteins as predictive factors to identify patients with high risk of cell transformation and HCC development

Loss of the Orphan Nuclear Receptor SHP Is More Pronounced in Fibrolamellar Carcinoma than in Typical Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) remains a major problem in oncology. The molecular mechanisms which underlie its pathogenesis are poorly understood. Recently the Small Heterodimer Partner (SHP), an orphan nuclear receptor, was suggested to be involved as a tumor suppressor in hepatocellular carcinoma development. To date, there are no such studies regarding fibrolamellar carcinoma, a less common variant of HCC, which usually affects young people and displays distinct morphological features. The aim of our project was to evaluate the SHP levels in typical and fibrolamellar hepatocellular carcinoma with respect to the levels of one of the cell cycle regulators, cyclin D1. We assessed the immunoreactivity levels of SHP and cyclin D1 in 48 typical hepatocellular carcinomas, 9 tumors representing the fibrolamellar variant, 29 non malignant liver tissues and 7 macroregenerative nodules. We detected significantly lower SHP immunoreactivity in hepatocellular carcinoma when compared to non malignant liver tissue. Moreover, we found that SHP immunoreactivity is reduced in fibrolamellar carcinoma when compared to typical hepatocellular carcinoma. We also found that SHP is more commonly lost in HCC which arises in the liver with steatosis. The comparison between the cyclin D1 and SHP expression revealed the negative correlation between these proteins in the high grade HCC. Our results indicate that the impact of loss of SHP protein may be even more pronounced in fibrolamellar carcinoma than in a typical form of HCC. Further investigation of mechanisms through which the loss of SHP function may influence HCC formation may provide important information in order to design more effective HCC therapy