Although deregulated expression of specific microRNAs (miRNAs) has been described in solid cancers and leukemias, little evidence of miRNA deregulation has been reported in ALK-positive (ALK ؉ ) anaplastic large cell lymphomas (ALCL). These tumors overexpress the major antiapoptotic protein myeloid cell leukemia 1 (MCL-1), a situation that could compensate for the lack of BCL-2. We report that ALK ؉ ALCL cell lines and biopsy specimens (n ‫؍‬ 20) express a low level of miR-29a and that this down- IntroductionAnaplastic lymphoma kinase-positive (ALK ϩ ) anaplastic large cell lymphoma (ALCL) is now recognized as a distinct entity in the World Health Organization (WHO) classification of hematopoietic tumors 1,2 and is characterized by the expression of an oncogenic fusion protein involving the ALK tyrosine kinase receptor. 3,4 Most of the activated pathways downstream to this fusion protein with a constitutive tyrosine kinase activity have been characterized and play major roles in lymphomagenesis in ALK ϩ ALCL, controlling key cellular processes such as proliferation, survival, and cell migration (for review see Chiarle et al 5 ). Another characteristic of ALK ϩ ALCLs is the lack or low expression of the antiapoptotic proteins BCL-2 and BCL-XL, suggesting a possible explanation for their relatively good prognosis. However, these tumors overexpressed MCL-1 (myeloid cell leukemia-1), an oncogene of particular interest, belonging to BCL-2 family of apoptosisregulating proteins, 6 and also involved in programming differentiation 7 and promoting cell viability. 8 MCL-1 expression could compensate for the lack of immunohistochemically detectable BCL-2 and BCL-XL expression in ALK ϩ ALCL. [9][10][11] Some studies have suggested that the Jak-STAT and PI3K pathways activated in ALK ϩ tumors could be involved in up-regulating MCL-1 but other pathways at the posttranscriptional level, such as microRNAs, might also contribute to its high expression in ALK ϩ ALCL cases (see reviews by Akgul 12 and Michels et al 13 ).Micro-RNAs (miRNAs) are small noncoding RNAs that regulate target gene expression posttranscriptionally through base pairing within the 3Ј-UTR regions of the target messenger RNAs and inducing their degradation, translational inhibition, or both of the encoded proteins. 14,15 MiRNAs play key regulator roles in fundamental biologic processes including cell differentiation, apoptosis, cell proliferation, organ development, and hematopoiesis (see review by Kluiver et al 16 ). family members have been shown to be down-regulated in several hematopoietic neoplasms, including chronic lymphocytic leukemia with poor prognosis, 17 acute myeloid leukemia, 18 and mantle cell lymphoma, 19 as well as solid cancers such as lung cancer, 20 hepatocellular carcinoma, 21 and invasive breast cancer. 22 More particularly, miR-29a, miR-29b, or both directly target the antiapoptotic protein MCL-1 in cholangiocarcinoma, 23 hepatocellular carcinoma, 21 and acute myeloid leukemia (AML). 18 However, to date, only one study has addressed th...

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Following computer searches of sequence banks, we have positively identified a novel intronic snoRNA, U24, encoded in the ribosomal protein L7a gene in humans and chicken. Like previously reported intronic snoRNAs, U24 is devoid of a 5'-trimethyl-cap. U24 is immunoprecipitated by an antifibrillarin antibody and displays an exclusively nucleolar localization by fluorescence microscopy after in situ hybridization with antisense oligonucleotides. In vertebrates, U24 is a 76 nt long conserved RNA which is metabolically stable, present at approximately 14,000 molecules per human HeLa cell. U24 exhibits a 5'-3' terminal stem-box C-box D structure, typical for several snoRNAs, and contains two 12 nt long conserved sequences complementary to 28S rRNA. It is, therefore, strikingly related to U14, U20 and U21 snoRNAs which also possess long sequences complementary to conserved sequences of mature 18S or 28S rRNAs. In 28S rRNA the two tracts complementary to U24 are adjacent to each other, they involve several methylated nucleotides and are surprisingly close, within the rRNA secondary structure, to complementarities to snoRNAs U18 and U21. Identification of the yeast Saccharomyces cerevisiae U24 gene directly confirms the outstanding conservation of the complementarity to 28S rRNA during evolution, suggesting a key role of U24 pairing to pre-rRNA during ribosome biogenesis, possible in the control of pre-rRNA folding. Yeast S.cerevisiae U24 is also intron-encoded but not in the same host-gene as in humans or chicken.

We identified the first archaeal tRNA ribose 2 0 -O-methylase, aTrm56, belonging to the Cluster of Orthologous Groups (COG) 1303 that contains archaeal genes only. The corresponding protein exhibits a SPOUT S-adenosylmethionine (AdoMet)-dependent methyltransferase domain found in bacterial and yeast G18 tRNA 2 0 -O-methylases (SpoU, Trm3). We cloned the Pyrococcus abyssi PAB1040 gene belonging to this COG, expressed and purified the corresponding protein, and showed that in vitro, it specifically catalyzes the AdoMet-dependent 2 0 -O-ribose methylation of C at position 56 in tRNA transcripts. This tRNA methylation is present only in archaea, and the gene for this enzyme is present in all the archaeal genomes sequenced up to now, except in the crenarchaeon Pyrobaculum aerophilum. In this archaea, the C56 2 0 -O-methylation is provided by a C/D sRNP. Our work is the first demonstration that, within the same kingdom, two different mechanisms are used to modify the same nucleoside in tRNAs.

Following a search of sequence data bases for intronic sequences exhibiting structural features typical of snoRNAs, we have positively identified by Northern assays and sequence analysis another intron-encoded snoRNA, termed U21. U21 RNA is a 93 nt. long, metabolically stable RNA, present at about 10(4) molecules per HeLa cell. It is encoded in intron 5 of the ribosomal protein L5 gene, both in chicken and in the two mammals studied so far, human and mouse. U21 RNA is devoid of a 5'-trimethyl-cap and is likely to result from processing of intronic RNA. The nucleolar localization of U21 has been established by fluorescence microscopy after in situ hybridization with digoxigenin-labeled oligonucleotide probes. Like most other snoRNAs U21 contains the box C and box D motifs and is precipitated by anti-fibrillarin antibodies. By the presence of a typical 5'-3' terminal stem, U21 appears more particularly related to U14, U15, U16 and U20 intron-encoded snoRNAs. Remarkably, U21 contains a long stretch (13 nt.) of complementarity to a highly conserved sequence in 28S rRNA. Sequence comparisons between chicken and mammals, together with Northern hybridizations with antisense oligonucleotides on cellular RNAs from more distant vertebrates, point to the preferential preservation of this segment of U21 sequence during evolution. Accordingly, this complementarity, which overlaps the complementarity of 28S rRNA to another snoRNA, U18, could reflect an important role of U21 snoRNA in the biogenesis of large ribosomal subunit.

Recently, several new snoRNAs encoded in introns of genes coding for ribosomal, ribosome-associated, or nucleolar proteins have been discovered. We are presently studying four of these intronic snoRNAs. Three of them, U20, U21, and U24, are closely related to each other on a structural basis. They are included in genes encoding nucleolin and ribosomal proteins L5 and L7a, respectively, in warm-blooded vertebrates. These three metabolically stable snoRNAs interact with nucleolar protein fibrillarin. In addition, they display common features that make them strikingly related to snoRNA U14. U14 contains two tracts of complementarity to 18S rRNA, which are required for the production of 18S rRNA. U20 displays a 21 nucleotide (nt) long complementarity to 18S rRNA. U21 contains a 13 nt complementarity to an invariant sequence in eukaryotic 28S rRNA. U24 has two separate 12 nt long complementarities to a highly conserved tract of 28S rRNA. Phylogenetic evidences support the fundamental importance of the pairings of these three snoRNAs to pre-rRNA, which could be involved in a control of pre-rRNA folding during preribosome assembly. By transfection of mouse cells, we have also analyzed the processing of U20 and found that the -cis acting signals for its processing from intronic RNA are restricted to the mature snoRNA sequence. Finally, we have documented changes of host genes for these three intronic snoRNAs during the evolution of eukaryotes.

We report the complete nudeotide sequence of the 3627 bp long Y-external transcribed spacer (ETS) of a human ribosomal RNA gene. This sequence exhibits only very limited homologies with its mouse counterpart, the only other mammalian specimen analyzed so far. It has very peculiar compositional characteristics, with a highly biased base content (very rich in G + C, very poor in A) and also some very strong dinucleotide preferences. Interestingly, these specific features are shared by the mouse sequence, despite the extensive sequence divergence, and also apply to the other transcribed spacers of mammals indicating that a common and strong structural constraint is exerted on all these regions of the ribosomal gene. An outstanding secondary structure can be formed within the human ETS RNA, which could have a significant role in preribosome assembly.

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