The mouse Krüppel-like Factor 4 (Klf4) gene: Four functional polyadenylation sites which are used in a cell-specific manner as revealed by testicular transcript analysis and multiple processed pseudogenes

The transcription factor Krüppel-like factor 4 (Klf4) is involved in cell cycle arrest and terminal differentiation of many epithelial cell types. We have recently shown that Northern blot analysis of RNA from adult mouse testis revealed multiple Klf4 transcripts. In order to characterize these transcripts, we tested for alternative splicing events and looked for alternative transcriptional initiation and usage of different polyadenylation signals. We neither obtained evidence for alternative splicing nor found transcripts with novel 5′ ends. However, we found striking differences in the 3′ ends by RACE-PCR. These differences were, interestingly, due to the usage of four alternatively used polyadenylation signals (PAS). This high number of PAS is found in less than 1% of all genes. We show that testicular Sertoli cells exclusively use the first PAS, which is, notably, not canonical, while haploid germ cells rather use the more 3′ located PAS-II-IV. The longer transcripts present in germ cells exhibit highly conserved putative binding motifs for proteins known to be important for translational regulation in germ cells. Moreover, we experimentally confirm an intron which was not described in a previous report on the Klf4 gene structure. Finally, we document six Klf4 pseudogenes most likely formed by L1-mediated retrotransposition, indicating germ line expression of Klf4. In summary, we show that mouse testicular cells make intensive use of alternative polyadenylation of Klf4 mRNA strongly suggesting translational regulation of the Klf4 message in spermatids.