Cascaded multiple classifiers for secondary structure prediction

We describe a new classifier for protein secondary structure prediction that is formed by cascading together different types of classifiers using neural networks and linear discrimination. The new classifier achieves an accuracy of 76.7% (assessed by a rigorous full Jack-knife procedure) on a new nonredundant dataset of 496 nonhomologous sequences (obtained from G.J. Barton and J.A. Cuff). This database was especially designed to train and test protein secondary structure prediction methods, and it uses a more stringent definition of homologous sequence than in previous studies. We show that it is possible to design classifiers that can highly discriminate the three classes (H, E, C) with an accuracy of up to 78% for beta-strands, using only a local window and resampling techniques. This indicates that the importance of long-range interactions for the prediction of beta-strands has been probably previously overestimated.     

Specific Tandem 3'UTR Patterns and Gene Expression Profiles in Mouse Thy1+ Germline Stem Cells

A recently developed strategy of sequencing alternative polyadenylation (APA) sites (SAPAS) with second-generation sequencing technology can be used to explore complete genome-wide patterns of tandem APA sites and global gene expression profiles. spermatogonial stem cells (SSCs) maintain long-term reproductive abilities in male mammals. The detailed mechanisms by which SSCs self-renew and generate mature spermatozoa are not clear. To understand the specific alternative polyadenylation pattern and global gene expression profile of male germline stem cells (GSCs, mainly referred to SSCs here), we isolated and purified mouse Thy1⁺ cells from testis by magnetic-activated cell sorting (MACS) and then used the SAPAS method for analysis, using pluripotent embryonic stem cells (ESCs) and differentiated mouse embryonic fibroblast cells (MEFs) as controls. As a result, we obtained 99,944 poly(A) sites, approximately 40% of which were newly detected in our experiments. These poly(A) sites originated from three mouse cell types and covered 17,499 genes, including 831 long non-coding RNA (lncRNA) genes. We observed that GSCs tend to have shorter 3''UTR lengths while MEFs tend towards longer 3''UTR lengths. We also identified 1337 genes that were highly expressed in GSCs, and these genes were highly consistent with the functional characteristics of GSCs. Our detailed bioinformatics analysis identified APA site-switching events at 3''UTRs and many new specifically expressed genes in GSCs, which we experimentally confirmed. Furthermore, qRT-PCR was performed to validate several events of the 334 genes with distal-to-proximal poly(A) switch in GSCs. Consistently APA reporter assay confirmed the total 3''UTR shortening in GSCs compared to MEFs. We also analyzed the cis elements around the proximal poly(A) site preferentially used in GSCs and found C-rich elements may contribute to this regulation. Overall, our results identified the expression level and polyadenylation site profiles and these data provide new insights into the processes potentially involved in the GSC life cycle and spermatogenesis.     

Genome level analysis of rice mRNA 3'-end processing signals and alternative polyadenylation

The position of a poly(A) site of eukaryotic mRNA is determined by sequence signals in pre-mRNA and a group of polyadenylation factors. To reveal rice poly(A) signals at a genome level, we constructed a dataset of 55 742 authenticated poly(A) sites and characterized the poly(A) signals. This resulted in identifying the typical tripartite cis-elements, including FUE, NUE and CE, as previously observed in Arabidopsis. The average size of the 3′-UTR was 289 nucleotides. When mapped to the genome, however, 15% of these poly(A) sites were found to be located in the currently annotated intergenic regions. Moreover, an extensive alternative polyadenylation profile was evident where 50% of the genes analyzed had more than one unique poly(A) site (excluding microheterogeneity sites), and 13% had four or more poly(A) sites. About 4% of the analyzed genes possessed alternative poly(A) sites at their introns, 5′-UTRs, or protein coding regions. The authenticity of these alternative poly(A) sites was partially confirmed using MPSS data. Analysis of nucleotide profile and signal patterns indicated that there may be a different set of poly(A) signals for those poly(A) sites found in the coding regions. Based on the features of rice poly(A) signals, an updated algorithm termed PASS-Rice was designed to predict poly(A) sites.     

Genome-Wide Control of Polyadenylation Site Choice by CPSF30 in Arabidopsis

The Arabidopsis thaliana ortholog of the 30-kD subunit of the mammalian Cleavage and Polyadenylation Specificity Factor (CPSF30) has been implicated in the responses of plants to oxidative stress, suggesting a role for alternative polyadenylation. To better understand this, poly(A) site choice was studied in a mutant (oxt6) deficient in CPSF30 expression using a genome-scale approach. The results indicate that poly(A) site choice in a large majority of Arabidopsis genes is altered in the oxt6 mutant. A number of poly(A) sites were identified that are seen only in the wild type or oxt6 mutant. Interestingly, putative polyadenylation signals associated with sites that are seen only in the oxt6 mutant are decidedly different from the canonical plant polyadenylation signal, lacking the characteristic A-rich near-upstream element (where AAUAAA can be found); this suggests that CPSF30 functions in the handling of the near-upstream element. The sets of genes that possess sites seen only in the wild type or mutant were enriched for those involved in stress and defense responses, a result consistent with the properties of the oxt6 mutant. Taken together, these studies provide new insights into the mechanisms and consequences of CPSF30-mediated alternative polyadenylation.     

Knock-down of 25 kDa subunit of cleavage factor Im in Hela cells alters alternative polyadenylation within 3'-UTRs

Alternative polyadenylation leads to mRNAs with variable 3′ ends. Since a 3′-untranslated region (3′-UTR) often contains cis elements that impact stability or localization of mRNA or translation, selection of poly(A) sites in a 3′-UTR is regulated in mammalian cells. However, the molecular basis for alternative poly(A) site selection within a 3′-UTR has been unclear. Here we show involvement of cleavage factor Im (CFIm) in poly(A) site selection within a 3′-UTR. CFIm is a heterodimeric 3′ end-processing complex, which functions to assemble other processing factors on pre-mRNA in vitro. We knocked down 25 kDa subunit of CFIm (CFIm25) in HeLa cells and analyzed alternative poly(A) site selection of TIMP-2, syndecan2, ERCC6 and DHFR genes by northern blotting. We observed changes in the distribution of mRNAs in CFIm25 depleted cells, suggesting a role for CFIm in alternative poly(A) site selection. Furthermore, tissue specific analysis demonstrated that the CFIm25 gene gave rise to 1.1, 2.0 and 4.6 kb mRNAs. The 4.6 kb mRNA was ubiquitously expressed, while the 1.1 and 2.0 kb mRNAs were expressed in a tissue specific manner. We found three likely poly(A) sites in the CFIm25 3′-UTR, suggesting alternative polyadenylation. Our results indicate that alternative poly(A) site selection is a well-regulated process in vivo.     

Alternative Polyadenylation:Methods,Findings,and Impacts

Alternative polyadenylation(APA), a phenomenon that RNA molecules with different 30 ends originate from distinct polyadenylation sites of a single gene, is emerging as a mechanism widely used to regulate gene expression. In the present review, we first summarized various methods prevalently adopted in APA study, mainly focused on the next-generation sequencing(NGS)-based techniques specially designed for APA identification, the related bioinformatics methods, and the strategies for APA study in single cells. Then we summarized the main findings and advances so far based on these methods, including the preferences of alternative poly A(pA) site, the biological processes involved, and the corresponding consequences. We especially categorized the APA changes discovered so far and discussed their potential functions under given conditions, along with the possible underlying molecular mechanisms. With more in-depth studies on extensive samples,more signatures and functions of APA will be revealed, and its diverse roles will gradually heave in sight.