For the analysis of YTHDF2 messenger RNA expression in different tissues, the Human MTC Panel I and Human MTC Panel II were used. They consist of panels of first-strand complementary DNA preparations from different tissues and/or organs, pooled from different individuals.ages 20-28 years; small intestine 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 corresponding chromosomal positions) are referred to as the Human July 2003-hg16 assembly, available at the UCSC website. The detailed analysis of repetitive element composition of the genomic sequence corresponding to the QM376-400 DNA fragment and to the surrounding portion of the YTHDF2 fourth intron (chr. 1: 28,773,429-28,775,861) was performed using the Censor program (27) and the Repbase database of repetitive elements (28). The Alu density in a 2.6 Mb genomic region including the YTHDF2 gene (chr. 1: 27,500,001-30,100,000) was calculated in 100 kb intervals by means of the Repeat Masker program (http://www.repeatmasker.org). Gene density was calculated in a 1.6 Mb region (chr. 1: 27,600,002-29,200,000) based on the RefSeq Genes track (29), available at the UCSC website. To evaluate the Alu density in the whole genome, data about the position and length of all the Alu sequences identified in the human genome were downloaded from the UCSC website (Repeat Masker analysis on the Jul 2003 assembly) and analyzed. The Alu density was calculated in non-overlapping 1 Mb windows on each chromosome. Intervals including more than 0.4 Mb of sequencing gaps were not considered in the analysis. Primers for the specific amplification of the YTHDF2 intron 4 microsatellite polymorphic locus were YTHDF2 I.F. (5'-GAAGGACCTATCAGAGGCAGTTTT-3') and YTHDF2 I.R. (5'-TTGCAGTGAGCCAAGATA-3'), designed using Primer3 software (30). The first primer was labeled with HEX (hexachlorofluorescein) fluorochrome. PCR reagents and buffers were: 10 pmol each primer, 200 uM each deoxynucleotide, 50 mM KCl, 10 mM Tris HCl pH 8.3, 1.5 mM Mg2+, 1 unit of Taq DNA polymerase (Eppendorf). PCR was performed on an Applied Biosystems 9700 thermal cycler, with predenaturation at 94°C for 4 minutes and 33 amplification cycles as follows: 94°C for 45 seconds; 60°C for 45 seconds; 72°C for 40 seconds; and final extension at 72°C for 7 minutes. PCR products were separated and analyzed by capillary electrophoresis on an ABI/Prism 310 Genetic Analyzer (Applied Biosystems). Six PCR fragments resulting from the amplification of different genomic DNA samples were sequenced, using the same protocols and materials reported above. YTHDF2 I.F. and YTHDF2 I.R. were respectively used as primers for sequencing reactions. A comparative Ct (threshold cycle) method was used to determine gene expression (31,32). In the real-time PCR assay on cDNA obtained from immortalized lymphocytes, the relative abundance of YTHDF2 cDNA versus the cDNA of the housekeeping gene ACTB was expressed as [(cDNA)YTHDF2/(cDNA)ACTB] ratio of each sample, normalized by the [(cDNA)YTHDF2/(cDNA)ACTB] ratio of a control sample (calibrator). The normalized ratio was calculated taking into account the different PCR efficiencies for target and reference amplification (32). For cDNA samples from human tissues (MTC Panels I and II), two housekeeping genes were used: ACTB and GAPD. For each tissue obtained, two different values for YTHDF2 expression were normalized against ACTB or GAPD, respectively: YTHDF2 expression normalized against that of the two housekeeping genes was given by the geometric mean of the two values in each sample (33). Intra-assay and inter-assay coefficients of variation were 16.1% and 24.4%, calculated repeating some samples in the same assay and in different assays, respectively. Reverse Transcription and Real-Time PCR The total RNA of immortalized lymphocytes derived from 51 participants (35 centenarians and 16 young individuals) was reverse transcribed with M-MLV reverse transcriptase (Ambion,As expected, the cloned QM376-400 fragment is flanked by two Alu sequences positioned in inverted orientation: an Alu Sx of 302 bp and an Alu Y of 278 bp. The tail of Alu Y is followed by the (TG)n microsatellite. The remaining part of the sequence consists of a 290 bp portion of a MER element (medium reiterated frequency repeat) (34,35) that, based on the comparison with the Repbase database of repetitive elements (28), is a Golem-B/MER7B type DNA nonautonomous transposon. The region surrounding the locus is almost entirely constituted, for roughly 2.5 kb, by three fragments of a MER transposon separated by intervening Alu elements. The reciprocal positions of these repetitive elements could be explained assuming that, in the evolutionary history of the locus, an ancient MER transposon became the target for successive insertions of new elements.

The results showed that the gene is included in a 1.6 Mb region (Figure 1, chr. 1: 27,600,001-29,200,000) with a very high Alu density (ranging from 27% to 51%). This 1.6 Mb Alu-rich domain is also characterized by high gene density, resulting in 61.4% being made up of exons and introns (the genomic average is 34.3%), with relatively short intergenic sequences. In particular, the 500 kb region (chr. 1: 28,517,396-29,017,396) surrounding YTHDF2 contains nine different protein coding genes (CHC1, SECP43, AF277181, MGC45806, TAF12, GMEB1, YTHDF2, OPRD1, EPB41), most of which are putatively involved in signal transduction, gene expression regulation, and cell division.