ure study of the physical and functional connections among posttranscriptional trans acting factors operating upon the p16 mRNA and other transcripts promises to be a particularly exciting endeavor. For instance, the co-transcriptional loading of trans factors can link transcriptional events with subsequent splicing, transport, stability, and translation, trans factors are implicated in mRNA stability and translation decisions made at P-bodies and other cytoplasmic structures, and are anticipated to interact richly upon the target mRNA. 23863710 For genes playing pivotal cellular functions, such as p16 and other tumor suppressors and senescenceassociated proteins, such multi-leveled, complex regulatory networks are expected. The resulting system of checks and Prediction of p16 mRNA as a target of miR-24 miR-24 Blocks p16 Translation bases in heteroduplex = 14; maximum folding energy for heteroduplex. buffer and the IP material was resolved by 14% SDS-PAGE, transferred onto PVDF membranes, and visualized and quantified using a PhosphorImager. Polyribosome Fractionation Cells were incubated with cycloheximide and cytoplasmic lysates were fractionated by centrifugation through 1050% linear sucrose gradients and divided into 10 fractions for analysis, as described. Supporting Information Found at: doi:10.1371/journal.pone.0001864.s001 RNA Isolation and analysis Total RNA was isolated with the Trizol reagent. Conventional and quantitative RT-PCR were done as described. Briefly, 1 mg of total RNA or 50% of RNA isolated from each gradient fraction were reverse transcribed using random hexamers and SSII RT; the resulting cDNA was amplified by PCR using gene-specific primer pairs; mature miR-24, 5S rRNA, and U6 snRNA were quantified using mirVana miRNA primer sets and qRT-PCR miRNA detection kit. MicroRNA microarray analysis was performed by using the miRCURYTM LNA Array microRNA Profile Service from Exiqon. Total RNA prepared from Y and S was used. Three independently prepared sample sets were used for microarray analysis; miRNAs were considered to be differentially expressed when they were upregulated or downregulated in at least two experiments. Found at: doi:10.1371/journal.pone.0001864.s002 Found at: doi:10.1371/journal.pone.0001864.s003 Found at: doi:10.1371/journal.pone.0001864.s004 Found at: doi:10.1371/journal.pone.0001864.s005 Found at: doi:10.1371/journal.pone.0001864.s006 Western Blotting Proteins were resolved by 14% SDS-PAGE and transferred to PVDF membranes. Primary antibodies recognized p16, a-tubulin, EGFP or b-actin; after secondary antibody incubations, protein bands were detected by ECL-plus. Found at: doi:10.1371/journal.pone.0001864.s007 Acknowledgments We thank C.-Y. Chen for providing reagents for these studies. Analysis of Nascent Protein De novo p16 or GAPDH protein synthesis was measured by incubating HeLa cells with 1 mCi L-methionine and Cilomilast chemical information Lcysteine 16103101 for 15 min, followed by lysis with RIPA buffer and 
IP with 10 mg anti-p16 or anti-GAPDH antibodies; IgG was used in control IP reactions. Beads were washed in RIPA In eukaryotes, DNA is packaged into highly structured chromatin filaments, which are further compacted into chromosomes. The structure of eukaryotic chromatin and chromosomes influences accessibility of proteins and multi-protein machines to eukaryotic DNA, thereby influencing the efficiency and timing of DNA metabolic processes such as transcription, replication, DNA repair and recombination. Post-translational modificat
