BIOLOGICAL OVERVIEW

Mutations in pitchoune (pit) produce larvae that cannot grow beyond the first instar larval stage although they can live as long as 7-10 days. The name Pitchoune (meaning 'small' in the Provençal language) was chosen because Pit is a potential target of the dmyc gene, also termed diminutive. Analysis of mutant somatic clones suggests a function in cell growth and proliferation, which is supported by the fact that cell proliferation is promoted by pit overexpression. pit encodes a DEAD-box RNA helicase, a member of family of proteins involved in the control of RNA structure in many cellular processes. The closest homolog for this pit DEAD-box RNA helicase is a human DEAD-box RNA helicase, MrDb, whose corresponding gene transcription is directly activated by Myc-Max heterodimers. The discovery of pitchoune suggests that myc might promote cell proliferation by activating genes that are required in protein biosynthesis, thus linking cell growth and cell proliferation (Zaffran, 1998).

pitchoune was identifed by plasmid rescue of an enhancer trap line (1A122) whose reporter gene is expressed in all myogenic cells (Perrimon, 1991 and Zaffran, 1997). Screening of a genomic library led to the isolation of a lambda phage clone containing a 15 kb insert that was used to screen cDNA libraries. Two different classes of cDNAs were isolated. One of them corresponds to the held out wings (how) gene (Zaffran, 1997). The other transcription unit corresponds to pitchoune (Zaffran, 1998).

In the absence of pitchoune, larval tissues are no longer able to grow and their cells do not undergo the normal number of rounds of endoreplications. In the pit mutant, imaginal diploid cells, which in the wild type proliferate extensively during larval life and give rise to most of the adult structures, stop dividing very early in the larval stages. All tissues appear equally affected, in a precisely coordinated manner, and growth is arrested in the whole organism. pit expression appears ubiquitous, although to somewhat different extents, in all the investigated larval tissues. This suggests that the effect of the pit mutation is cell or tissue autonomous. However, the harmonious development of pit larvae is consistent with the existence of some kind of general signal that informs the different tissues to either continue their growth or to stop. Other results support the idea that pit participates in protein biosynthesis by controlling some aspect of the protein synthetic pathway: (1) the phenotype of the pit mutation is very reminiscent of homozygous Minute mutations, and Minute genes are known to encode ribosomal proteins; (2) the subcellular localization of pit is consistent with a function in the nucleolus, an organelle in which ribosome biogenesis takes place; and, (3) the proteins most closely related to Pit are DEAD-box RNA helicases, which are important players in rRNA processing or in ribosome biogenesis and which are also required for cell growth (Zaffran, 1998).

The DEAD-box RNA helicases family includes many members belonging to a wide spectrum of organisms ranging from bacteria to man. The DEAD-box RNA helicases have been proposed as major players in the modulations of RNA structure that provide an important means of regulating RNA function or accessibility in many physiological processes. Their property of unwinding RNA homoduplexes or RNA-DNA heteroduplexes can be utilized in rRNA processing, biogenesis of the ribosome, translation, mRNA splicing, and transcription among other biological functions. However, RNA helicase activity has been formally demonstrated for only a limited number of members such as eIF-4A, p68, An3 and Vasa: it has been assumed to be the case for the other members, based on sequence comparisons. The best characterized DEAD-box protein, eIF-4A, is a translation initiation factor required for cap-stimulated 40S ribosomal subunit recruitment. At least four yeast helicases are believed to participate in the formation of the spliceosome and in mRNA splicing. A human helicase regulates transcription by directly recruiting the CBP protein to RNA polymerase II. A Drosophila helicase may be active in the opening of chromatin structure (Eberl, 1997), as evidenced by the fact that mutations in this gene enhance position-effect variegation. Some RNA helicases may participate in a specific developmental event or be active only in a given tissue, especially in the germline. For example, Vasa is required for the formation of the germ cells. This representative, but by no means exhaustive, list of the processes in which RNA helicases might be involved illustrates the difficulties encountered in assigning a precise function to Pit on the sole basis of its structural homologies. However, the subcellular localization of Pit to the nucleolus suggests a role in this organelle. Indeed, several RNA helicases from other organisms have also been shown to be present in the nucleolus and their function in ribosome biogenesis or in rRNA processing has been well documented (Zaffran, 1998 and references).

In conclusion, it seems very likely that Pit participates in protein biosynthesis, probably by allowing for the correct maturation and functionality of the ribosomes. The absence of Pit could decrease protein synthesis to a level so low that it would be insufficient to promote cell growth and consequently cell division and proliferation. The effects of overexpression of Pit in imaginal discs suggest a positive modification of growth rate, probably by acting as a general and rate-limiting factor in ribosome biosynthesis. Finally, the protein sequence closest to that of Pit is MrDb, a human DEAD-box RNA helicase. The gene encoding that protein has been identified as a direct target of Myc because Myc-Max heterodimers were isolated as structures bound to the MrDb gene (Grandori, 1996). A canonical sequence, CACGTG, responsible for the binding of Myc was found in the coding sequence for MrDb but not in the pit gene, at least not in the transcribed part. However, pit transcription can be activated by ectopically expressed d-Myc, which suggests that, in Drosophila, pit could also be a potential target for Myc (Zaffran, 1998).

GENE STRUCTURE

Bases in 5' UTR - 62+

Exons - 3 or more

Bases in 3' UTR - 112

PROTEIN STRUCTURE

Amino Acids - 672

Structural Domains

The pit gene encodes an RNA helicase with an unusual DEAD-box (DEVD), homologous to human MrDb, whose gene is a direct target of Myc. Searches in sequences database libraries reveal that the deduced Pit protein sequence displays a significant homology score when compared with members of the DEAD-box family of ATPase RNA-dependent helicases that have been implicated in diverse cellular functions such as RNA splicing, ribosome assembly, initiation of translation and stabilization of RNA. In particular, the Pit-encoded protein contains in its core region the eight motifs with strong sequence conservation that are hallmarks of the family including the ATPase A and B motifs, the SAT motif and the HRIGR region. In Pit, however, the alanine in DEAD is replaced by a valine (DEVD). Another ATPase RNA-dependent helicase from Drosophila, Helicase at 25E, unambiguously belonging to the DEAD-box family, and has been reported to contain a DECD sequence (Eberl, 1997). Other RNA helicases in Drosophila include Homeless, Vasa and Maleless. Homeless, Vasa and Maleless belong to distinctively different sub-branches of the RNA helicase than that occupied by Pit (Zaffran, 1998).

The N-terminal domain (1 to 175 aa) of Pit has a very polar structure rich in lysine and Asp-Glu residues, which suggests a relationship, although only distantly, to the nucleolin family. The general organization of this N-terminal domain also shares important overall similarities with the nucleolar P68 protein. A bipartite nuclear localization signal is found in the N-terminal part of Pit, suggesting that this protein exerts its function in the nucleus. Interestingly, Pit shares very important similarities with human MrDb that extend over the entire length of the two proteins (Grandori, 1996; 59% identities) and with two predicted yeast RNA helicases of unknown function. The human MrDb RNA helicase was isolated as being a direct Myc-Max heterodimers target gene (Grandori, 1996). An even higher level of homology is observed when only the helicase domain (77% identity over 500 amino acids) is considered. Moreover, the conservation between these two proteins extends outside the helicase region, since the N-terminal domains of both proteins share a poly(K) stretch and a D/E-rich region. This analysis suggests that Pit, MrDb and the yeast putative RNA helicases could define a new subgroup within the DEAD-box family that is clearly apparent from the evolutionary tree (Zaffran, 1998).

EVOLUTIONARY HOMOLOGS

Information about the roles of RNA helicases in transcription and translation can be found at the Homeless, Vasa and Maleless sites.

The c-Myc protein is involved in cell proliferation, differentiation and apoptosis though heterodimerization with Max to form a transcriptionally active sequence-specific DNA binding complex. By means of sequential immunoprecipitation of chromatin using anti-Max and anti-Myc antibodies, a Myc-regulated gene and genomic sites occupied by Myc-Max in vivo have been identified. Four of 27 sites recovered by this procedure correspond to the highest affinity 'canonical' CACGTG sequence. However, the most common in vivo binding sites belong to the group of 'non-canonical' E box-related binding sites previously identified by in vitro selection. Several of the genomic fragments isolated contain transcribed sequences, including one, MrDb, encoding an evolutionarily conserved RNA helicase of the DEAD box family. The corresponding mRNA is induced following activation of a Myc-estrogen receptor fusion protein (Myc-ER) in the presence of a protein synthesis inhibitor, consistent with this helicase gene being a direct target of Myc-Max. As for c-Myc, the expression of MrDb is induced upon proliferative stimulation of primary human fibroblasts as well as B cells and down-regulated during terminal differentiation of HL60 leukemia cells. These results indicate that (1) Myc-Max heterodimers interact in vivo with a specific set of E box-related DNA sequences, and (2) that Myc is likely to activate multiple target genes, including a highly conserved DEAD box protein. Therefore, Myc may exert its effects on cell behavior through proteins that affect RNA structure and metabolism (Grandori, 1996).

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