La propiedad como derecho de protección

The heterogeneous ribonuclear proteins (hnRNPs) family is a class of diverse RNA- binding proteins that associate with nascent pre-mRNA. These factors remain associated with pre-mRNA until its processing is completed and with mRNAs during export from nucleus to cytoplasm (Izaurralde and Mattaj 1995).

Approximately >20 proteins have been identified by two-dimensional gel electrophoresis of human hnRNP complexes with molecular weight ranging from 34 (hnRNP A l) to 120 kDa (hnRNP U) (Dreyfuss, Matunis et al. 1993; Dreyfuss, Kim et al. 2002). Some hnRNPs are extremely abundant (-100 million copies per nucleus), while others are present in lower amount (Kamma, Portman et al. 1995;

Markovtsov, Nikolic et al. 2000). For many of these proteins multiple isoforms are produced by alternative splicing processes. This diversity is further increased by post-translational modifications of potential physiological significance, including phosphorylation, arginine methylation and SUMOylation (Dreyfuss, Kim et al. 2002;

Martinez-Contreras, Cloutier et al. 2007).

The structure of hnRNP proteins is modular and consists of one or more RNA binding domains associated with an auxiliary domain often involved in protein- protein interactions (Dreyfuss, Matunis et al. 1993). For instance, the hnRNP A/B proteins contain two RNP domains at the N-terminus and a Gly-rich auxiliary domain at the carboxy end. HnRNP E1-E2 proteins contain three KH domains (Ostareck-Lederer et al., 1998). The hnRNP H family members contain two (2H9) or three (H, H ’ and F) quasi RNA recognition motifs (qRRMs) and one or two glycine rich auxiliary domains (Honore, Rasmussen et al. 1995).

The hnRNP proteins show general RNA-binding specificity and individual proteins display preference for specific sequences that tend to coincide with sites of functional importance in pre-mRNA processing. However, hnRNP proteins generally do not bind specific sites exclusively but recognize different RNA sequences with a wide spectrum of affinities. This RNA binding ability is further modulated by cooperative protein-protein interactions (Dreyfuss, Matunis et al. 1993; Dreyfuss, Kim et al. 2002).

The hnRNP proteins frequently mediate splicing repression, particularly through binding to exonic splicing silencer elements or by sterical interference with other protein interaction (Cartegni, Chew et al. 2002). Nevertheless, depending on the position of the splicing regulatory elements hnRNPs can also associate with enhancer elements to help exon inclusion (Caputi and Zahler 2002) and a generic role for hnRNP A l and F/H proteins in intron definition has been recently proposed (Martinez-Contreras, Fisette et al. 2006).

Although many of the hnRNPs localized in the nucleus, a subset of these proteins shuttles continuously between nucleus and cytoplasm. This indicates a role of these proteins in nuclear export and in other cytoplasm processes (Pinol-Roma and Dreyfuss 1992; Martinez-Contreras, Cloutier et al. 2007).

hnRNP A l : Among the hnRNP proteins, the most abundant and extensively studied is hnRNP A l (Mayeda and Krainer 1992). hnRNP A l binds RNA through two RNA recognition motifs at its N terminus while the C-terminal domain comprises several RGG repeats, which also contribute to RNA binding. In vivo, four arginine residues within the RGG repeats of hnRNP A l are methylated and are thought to influence the RNA-binding properties (Kim, Merrill et al. 1997). The C-terminus also includes the M9 motif, involved in hnRNP A l nuclear import and export (Izaurralde, Jarmolowski et al. 1997).

Although at steady state hnRNP A l is predominantly nuclear, it shuttles rapidly between nuclear and cytoplasmic compartments (Pinol-Roma and Dreyfuss 1992).

The shuttling of hnRNP A l is subject to regulation and it has been proposed to have a role in cell proliferation, survival, and differentiation of normal and transformed cells (Iervolino, Santilli et al. 2002). The endogenous hnRNP A l is weakly phosphorylated in cells grown under normal conditions while upon osmotic shock a cytoplasmic accumulation of hnRNP A l is induced, concomitant with an increase in its phosphorylation state (van der Houven van Oordt, Diaz-Meco et al. 2000).

Since the early 1990s hnRNP A l has been associated with the regulation of alternative splicing process (Caceres and Komblihtt 2002). The first hnRNP A l- dependent ESS was identified in studies of HIV tat exon 2 regulation (Amendt, Hesslein et al. 1994; Amendt, Si et al. 1995). HnRNP A l splicing repression and the existence of Al-dependent ESSs have been documented in other numerous examples in humans (Del Gatto-Konczak, Olive et al. 1999; Kashima and Manley 2003;

Disset, Bourgeois et al. 2006).

Despite its extensive characterization only a small number of high affinity sites have been obtained by SELEX (Burd and Dreyfuss 1994). Interestingly a recent study, based on searching for endogenous hnRNP A l target RNAs, reported a specific

binding for the pri-miR-18a. This result highlighted a new role for hnRNP A l as auxiliary factor involved in miRNA processing (Guil and Caceres 2007).

Several mechanisms have been suggested to explain hnRNP A 1-mediated splicing repression. The hnRNP A l antagonizes in a concentration-dependent way the activity of SR proteins on the selection of 5’splice site and can bind to certain exon splicing silencers to prevent the use of adjacent 3’splice site (Mayeda and Krainer 1992; Smith and Valcarcel 2000). Another mechanism was elucidated studying the alternative splicing of the hnRNP A l transcript itself. The hnRNP A l protein can function as splicing repressor, influencing the alternative splicing of its own pre- mRNA, by binding to a conserved intronic element present in both introns surrounding the alternative exon 7b (Chabot, Blanchette et al. 1997; Blanchette and Chabot 1999; Hutchison, LeBel et al. 2002). Cooperation between the two A l complexes on these sites was suggested as promoting “ looping-out” of the intervening RNA, including exon 7b, thereby inhibiting splicing.

hnRNP A l is also implicated in various post-splicing activities, such as mRNA export (Izaurralde, Jarmolowski et al. 1997), mRNA stability (Hamilton, Bums et al.

1997) and cap-dependent and internal ribosome entry site-mediated translation (Svitkin, Ovchinnikov et al. 1996; Bonnal, Pileur et al. 2005). In addition it was reported that hnRNP A l has the ability to dismpt the higher order stmcture of telomeric DNA indicating a role in telomere maintenance (Zhang, Manche et al.

2006). HnRNP A l in fact binds with high affinity to telomeric single stranded DNA sequences (LaBranche, Dupuis et al. 1998; Dallaire, Dupuis et al. 2000) and interacts with telomerase RNA in vitro (Fiset and Chabot 2001). A severe hnRNP A l reduction in mouse erythroleukemic cells resulted in shortened telomeric repeats, which can be lengthened by restoring hnRNP A l expression (LaBranche, Dupuis et al. 1998).