We present a comprehensive map of over 1 million polyadenylation sites and quantify their usage in major cancers and tumor cell lines using direct RNA sequencing. We built the Expression and Polyadenylation Database to enable the visualization of the polyadenylation maps in various cancers and to facilitate the discovery of novel genes and gene isoforms that are potentially important to tumorigenesis. Analyses of polyadenylation sites indicate that a large fraction (∼30%) of mRNAs contain alternative polyadenylation sites in their 3′ untranslated regions, independent of the cell type. The shortest 3′ untranslated region isoforms are preferentially upregulated in cancer tissues, genome-wide. Candidate targets of alternative polyadenylation-mediated upregulation of short isoforms include POLR2K, and signaling cascades of cell–cell and cell–extracellular matrix contact, particularly involving regulators of Rho GTPases. Polyadenylation maps also helped to improve 3′ untranslated region annotations and identify candidate regulatory marks such as sequence motifs, H3K36Me3 and Pabpc1 that are isoform dependent and occur in a position-specific manner. In summary, these results highlight the need to go beyond monitoring only the cumulative transcript levels for a gene, to separately analysing the expression of its RNA isoforms.

Recently identified small (20 to 40 bases) RNAs, such as microRNAs (miRNAs) and endogenous small interfering RNAs (siRNAs) participate in important cellular pathways. In this report, we systematically characterized several novel features of human and viral RNA products smaller than miRNAs. We found that Kaposi sarcoma-associated herpesvirus K12-1 miRNA (23 bases) associates with a distinct, unusually small (17-base) RNA (usRNA) that can effectively downregulate a K12-1 miRNA target, human RAD21, suggesting that stable degradation-like products may also contribute to gene regulation. High-throughput sequencing reveals a diverse set of human miRNA-derived usRNAs and other non-miRNA-derived usRNAs. Human miRNA-derived usRNAs preferentially match to 5 ends of miRNAs and are also more likely to associate with the siRNA effector protein Ago2 than with Ago1. Many non-miRNA-derived usRNAs associate with Ago proteins and also frequently contain C-rich 3-specific motifs that are overrepresented in comparison to Piwi-interacting RNAs and transcription start site-associated RNAs. We postulate that approximately 30% of usRNAs could have evolved to participate in biological processes, including gene silencing.Recent studies have identified several classes of small RNAs, such as microRNAs (miRNAs), across a wide range of organisms (9). miRNAs can function as repressors (24) or activators (47) of gene expression. More than 500 human miRNAs are known, and many more are thought to exist (3). Advances in high-throughput sequencing have enabled the discovery of additional animal miRNAs and classes of small RNAs on an unprecedented scale across distantly related species, including animals, fungi, unicellular algae, plants, and viruses (9). In addition to miRNAs, recently identified metazoan small RNAs include endogenous small interfering RNAs (siRNAs) that mediate mRNA degradation (6,13,33,45), hairpin RNAs that are thought to repress target transcripts in flies (32), Piwi-interacting RNAs (piRNAs) that modulate spermatogenesis in mammals (14, 22), piRNA-like repeat-associated siRNAs that regulate chromatin structure of retrotransposons in insect germ lines (21), transcription start site-associated RNAs (TSSa-RNAs), and the functionally uncharacterized piRNA-like 21U-RNAs harboring 5Ј uridines in worms (36).The various small-RNA classes frequently share sequence characteristics and recruit similar protein partners. For example, the 5Ј ends of miRNAs (41, 49) and piRNAs (14) prefer uridines that likely bind to argonaute/Piwi proteins (44, 46). The argonaute (Ago) proteins bind miRNAs and siRNAs, while the Piwi proteins, a subgroup of the Ago family, bind piRNAs (9). Ago/Piwi-bound small RNAs are part of a larger ribonucleoprotein complex (RNP). The small RNAs in RNPs act as adaptors that recognize complementary nucleic acid targets and thus guide the RNPs to their target molecules (e.g., mRNAs) to direct processes, such as transcript cleavage (miRNAs, siRNAs, and piRNAs) and translational repression (miRNAs). Transcript cleavage by s...

This paper presents systematic investigation of the microchannel surface properties in microCE chips. Three popular materials for microCE chips, polydimethylsiloxane (PDMS), quartz, and glass, are used. The zeta potentials of these microchannels are calculated by measuring the EOF velocity to evaluate the surface properties after surface modification. The hydrophobic PDMS is usually plasma-treated for microCE applications. In this study, a new method using a high-throughput atmospheric plasma generator is adopted to treat the PDMS surface under atmospheric conditions. In this approach, the cost and time for surface treatment can be significantly reduced compared with the conventional vacuum plasma generator method. Experimental results indicate that new functional groups could be formed on the PDMS surface after treatment, resulting in a change in the surface property. The time-dependent surface property of the plasma-treated PDMS is then measured in terms of the zeta potential. Results show that the surface property will reach a stable condition after 1 h of plasma treatment. For glass CE chips, two new methods for changing the microchannel surface properties are developed. Instead of using complicated and time-consuming chemical silanization procedures for CE channel surface modification, two simple and reliable methods utilizing organic-based spin-on-glass and water-soluble acrylic resin are reported. The proposed method provides a fast batch process for controlling the surface properties of glass-based CE channels. The proposed methods are evaluated using PhiX-174 DNA maker separation. The experimental data show that the surface property is modified and separation efficiency greatly improved. In addition, the long-term stability of both coatings is verified in this study. The methods proposed in this study show potential as an excellent solution for glass-based microCE chip surface modification.

The highly conserved, multifunctional YB-1 is a powerful breast cancer prognostic indicator. We report on a pervasive role for YB-1 in which it associates with thousands of nonpolyadenylated short RNAs (shyRNAs) that are further processed into small RNAs (smyRNAs). Many of these RNAs have previously been identified as functional noncoding RNAs (http://www.johnlab.org/YB1). We identified a novel, abundant, 3′ -modified short RNA antisense to Dicer1 (Shad1) that colocalizes with YB-1 to P-bodies and stress granules. The expression of Shad1 was shown to correlate with that of YB-1 and whose inhibition leads to an increase in cell proliferation. Additionally, Shad1 influences the expression of additional prognostic markers of cancer progression such as DLX2 and IGFBP2. We propose that the examination of these noncoding RNAs could lead to better understanding of prostate cancer progression.

This work develops a double-sided probing system and calibration method for measuring the S-parameters of vertical interconnects at the wafer, package and socket level. The applicable device under test (DUTs) include through silicon vias (TSVs), plated through holes (PTHs), pogo pins and pressure sensitive conductive rubbers (PCRs). The effects of solder bumps and balls can also be taken into account. A short-open-load-reciprocal (SOLR) calibration method is used with a reciprocal thru to instead of the conventional shortopen-load-thru (SOLT) which uses a standard thru. The Sparameters can be measured up to 40 GHz with a repeatable S21 parameter accuracy of better than 0.2 dB and 1 degree in magnitude and phase, respectively. Additionally, the eye diagrams are measured at a maximum data rate of 40 Gb/s and a minimum rise time of 10 ps with the help of Agilent physical layer test system (PLTS).