M ETHODS

of the PCR assay were the following: primers R12A/267 labeled with tetrachlorofluorescein (TET), and R14B/264 labeled with 6-carboxyfluorescein (6-FAM); total reaction volume 50 ll, genomic DNA template 100 ng; 27 PCR cycles of 948C denaturation, 508C annealing, 728C exten- sion. Capillary electrophoresis (ABI/Prism 310; Applied Biosystems, Foster City, CA) evidenced, in the resulting inter-Alu pattern, the presence of a 384 bp fragment corresponding to an allele of the QM 376-400 poly- morphism. An aliquot of the PCR product was subjected to electrophoresis on a 1.5% agarose gel: The gel slice corresponding to an estimated length of 350–400 bp was cut, and the DNA was eluted and analyzed through capillary electrophoresis to confirm the presence and purity of the 384 bp fragment. An aliquot (0.1 ll) of the eluted DNA was used for a second cycle of PCR–gel electrophoresis–elution, aimed to increase the quantity of the specific PCR product. The purified DNA fragment was cloned in pPCR-Script plasmid and used to transform XL10-gold ultracompetent cells (Stratagene, La Jolla, CA). Cells were plated on Luria- Bertani agar plates containing S-gal (Sigma-Aldrich, St. Louis, MO), isopropyl b-D-thiogalactopyranoside (IPTG), and ampicillin for black–white color screening of recombi- nants; recombinant colonies were cultured in Luria-Bertani broth, and plasmidic DNA was extracted using the ‘‘miniprep’’ method (26). Plasmidic DNA extracted from 31 recombinant clones was used as the template for a PCR with R12A/267 and R14B/264 primers (same conditions previously indicated). Amplification products were digested with different restriction enzymes (NlaIII, AlwI, MspI, BstuI, DdeI, MseI, BstnI, and MnlI; provided by Celbio, Milano, Italy) and analyzed through agarose gel electrophoresis: The resulting restriction patterns were compared with the pattern generated by the same enzymes on an aliquot (4 lg) of the 384 bp DNA fragment used for the cloning. Four clones had DNA inserts of correct length (checked by capillary electrophoresis) and restriction pattern, and were selected for the sequencing. Sequencing reactions were conducted on 300 ng of plasmidic DNA from each selected clone and with M13 primer (59-TGTAAAACGACGGCCAGT-39), a ‘‘Big- Dye terminator reaction kit’’ (Applied Biosystems), and a 9700 thermal cycler (Applied Biosystems). Sequencing products were analyzed on an Abi/Prism 310 Genetic Analyzer (Applied Biosystems).

DNA and Complementary DNA Samples and B Lymphocyte Immortalization

of the Italian National Research Center on Aging (INRCA). Blood was collected by venipuncture after an informed con- sent form describing the project and its purpose had been explained to and signed by each recruited participant. Blood samples were obtained from a total of 412 unrelated partici- pants born and resident in Italy, belonging to two age groups: 275 controls (age 17–65 years, mean age 41 years; 152 men, 123 women) and 137 long-lived individuals (age 99–109 years, mean age 101 years; 26 men, 111 women), herein indicated as centenarians. The centenarians admitted to this study were in relatively good condition for their very ad- vanced age, even if some of them had decreased visual and auditory acuity. They did not have any major age-related diseases (severe cognitive or physical impairment, clinically evident cancer, or renal insufficiency) and belonged to cate- gory A and B of the centenarian health status classification (24). The younger control participants admitted to the study were free of any overt disease. DNA was extracted from blood samples following standard procedures. Peripheral blood mononuclear cells were obtained from 51 of the former samples, 16 young controls (mean age 36 years; 8 men, 8 women) and 35 centenarians (mean age 101 years; 4 men, 31 women). Peripheral blood mononuclear cell isolation, culture, and B-lymphocyte immortalization were performed as previously described (25). After the immortalization, the medium was substituted every 3 days and cells were harvested and frozen in liquid nitrogen 2 months later. For the analysis of YTHDF2 messenger RNA (mRNA) expression in different tissues, the ‘‘Human MTC Panel I’’ and ‘‘Human MTC Panel II’’ (BD Biosciences Clontech, Palo Alto, CA) were used. They consist of panels of first-strand complementary DNA (cDNA) preparations from different tissues and/or organs, pooled from different individuals. The detailed origin of each sample was the following: heart, pooled from 8 male/female Caucasians, age 25–59 years; brain (whole), from 2 male Caucasians, ages 43 and 55 years; placenta, from 7 Cauca- sians, ages 22–35 years; lung, from 2 female Caucasians, ages 24 and 32 years; liver, from 4 male/female Caucasians, ages 44–50 years; skeletal muscle, from 8 male/female Cauca- sians, ages 29–60 years; kidney, from 6 male/female Cauca- sians, ages 28–52 years; pancreas, from 20 male/female Caucasians, ages 25–59 years; spleen, from 11 male/female Caucasians, ages 24–70 years; thymus, from 18 male/fe- male Caucasians, ages 15–32 years; prostate, from 32 Cauca- sians,ages 21–50 years;testis, from45Caucasians, ages14–64 years; ovary, from 5 Caucasians, ages 20–28 years; small intes- tine without mucosal lining, from 32 male/female Caucasians, ages 15–57 years; colon with mucosal lining, from 20 male/ female Caucasians, ages 17–76 years; and leukocyte, peripheral blood, from 550 male/female Caucasians, ages 18–40 years.

Analysis of Alu Density and Gene Density