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THE CELL NUCLEUS AND RNA MOLECULES.

Fabio Cobianchi, Claudia Ghigna, Alessandra Montecucco, Silvano Riva e Giuseppe Biamonti

 

After the sequencing of the genome, the challenge today is to understand the molecular circuits that control gene expression in human cells. It is now clear that less than 5% of the human genome encodes for proteins. Nevertheless almost the entire genome is transcribed and most of the DNA sequences are in fact transcribed into non-coding (nc) RNAs. Although still poorly characterized, ncRNAs appear to exert regulatory functions in controlling gene expression at diverse levels.

 


Alternative splicing and tumor progression.

Claudia Ghigna, Giuseppe Biamonti

 


A significant fraction of ncRNAs is accounted for by introns that are interspersed with protein-coding exons in the primary gene transcripts. The reconstitution of an uninterrupted coding mRNA requires excision of introns that occurs with nucleotide precision during the splicing reaction. This is a challenging task for the splicing apparatus since exon/intron boundaries are characterized by very short consensus sequences. Moreover, introns are usually much longer than exons. A further level of complexity results from the fact that about 75% of the human genes undergo alternative splicing, i.e. the production of different mature mRNAs from pre-mRNAs encoded by a single gene. In this sense alternative splicing can be viewed as a mechanism to expand the coding capacity of the genome allowing the production of functionally distinct proteins by a single gene. Therefore, alternative splicing is potent mechanism to control gene expression at the prost-transcriptional level and significantly contributes to cell identity and to development.
Control of alternative splicing depends on a number of RNA binding proteins that are not stable components of the splicing machinery. These proteins interact in a sequence specific manner with the pre-mRNA molecule by binding to splicing regulatory sequence elements known as “enhancers” and “silencers” of splicing either in exons (ESE: Exonic Splicing Enhancer; ESS: Exonic Splicing Silencer) or in introns (ISE and ISS).

One goal of our research group is to understand how the altered expression and or activity of RNA binding proteins, that frequently occurs in cancers, may affect alternative splicing contributing to tumor progression. As a model we have studied the alternative splicing of the Ron proto-oncogene, which encodes for the membrane receptor of the cell scattering factor MSP (Macrophage Stimulating Protein). Interaction with MSP activates the tyrosine kinase activity of Ron which triggers a signal transduction cascades leading to cell motility, protection from apoptosis and reorganization of the cytoskeleton. This process, called “cell invasiveness” or “cell scattering”, is physiologically relevant during development and tissue regeneration and is a critical for the Epithelial to Mesenchimal cell Transition (EMT). EMT also occurs during tumor progression and it is responsible for the metastatic potential of most of human carcinomas.

 




Figure 1. Schematic representation of the portion of the Ron gene undergoing alternative splicing. Enhancer and Silencer element in exon 12 are indicated along with the interaction with splicing factor SF2/ASF. Bottom: Phase contrast images of human 293 cells transfected with an empty vector or with a plasmid that directs the over-expression of splicing factor SF2/ASF.


Skipping of exon 11 of the Ron pre-mRNA results in the production of a protein isoform, ΔRon, whose expression triggers EMT. A high level of ΔRon transcripts occurs in 75% of breast carcinomas. We have identified sequence elements and proteins controlling splicing of exon 11. Splicing factor SF2/ASF plays a critical role in the choice between inclusion and skipping of exon 11 and acts by binding to an ESE element located in exon 12. High levels of SF2/ASF, which frequently occurs in cancers, induce skipping of exon 11 and the production of ΔRon. We have shown that up-regulation of SF2/ASF is sufficient to trigger EMT and to promote cell motility. On the contrary, down regulation of SF2/ASF by RNA interference promotes MET (Mesenchimal to Epithelial cell Transition). The same effect is obtained by down-regulating the expression of ΔRon indicating that the effect on the alternative splicing of Ron transcripts is critical for the role of SF2/ASF in EMT.

Our studies provide the first clear evidence that the expression level of a specific splicing factor (SF2/ASF) is directly linked to tumor progression by affecting the splicing profile of a specific gene (Ron).

We are presently searching for additional factors that may contribute to control splicing of exon 11. We have already mapped a silencer in exon 12 (ESS) that counteract the activity of the ESE element bound by SF2/ASF.


References

  1. Ghigna C, Giordano S, Shen H, Benvenuto F, Castiglioni F, Comoglio PM, Green MR, Riva S, Biamonti G. (2005) Cell motility is controlled by SF2/ASF through alternative splicing of the Ron protooncogene. Mol Cell. 20:881-90.
  2. Shen H, Kan JL, Ghigna C, Biamonti G, Green MR. (2004) A single polypyrimidine tract binding protein (PTB) binding site mediates splicing inhibition at mouse IgM exons M1 and M2. RNA. 10:787-94.
  3. Ghigna C, Moroni M, Porta C, Riva S, Biamonti G. (1998) Altered expression of heterogenous nuclear ribonucleoproteins and SR factors in human colon adenocarcinomas. Cancer Res. 58:5818-24.


Non-coding RNAs, heterochromatin and structural/functional organization the human cell nucleus.

Giuseppe Biamonti

 


One of the most exciting findings concerns the pivotal role of ncRNAs in regulation of gene expression. Non-coding RNAs, in fact, have been shown to control different aspects of cell metabolism, such as chromatin organization, transcription and mRNA translation. The molecular details of these regulatory circuits are still largely to be characterized, however it is commonly accepted that short (20-25 nt long) RNA molecules, dubbed micro RNAs or miRNAs, play a central role. Studies in lower eukaryotes, such as Schizosaccharomyces pombe, have shown that miRNAs, originating from transcription of repetitive DNA sequences, direct the assembly of cognate DNA sequences into heterochromatin. Several data point to the existence of similar mechanisms in higher eukaryotes, including human. However, the situation is probably more complex in metazoan than in yeast, since RNA molecules larger than miRNAs, such as long RNAs involved in the X-inactivation process, are stable component of chromatin. The nature of these long ncRNAs is still matter of investigation.

Our laboratory has recently described a new class of long ncRNAs deriving from transcription of long tandem arrays of Satellite III repetitive DNAs found in pericentromeric heterochromatic portions of specific human chromosomes, such as chromosome 9. One strand (the G-rich strand) of the repeats is preferentially transcribed. A distinguishing feature of SatIII RNAs is the fact that these molecules are usually present at very low levels in the cells but their level dramatically increases (up to 10000 fold) after different types of stresses including heat shock, heavy metals, hyper-osmotic conditions and to a lesser extent UV light irradiation. Intriguingly, these ncRNAs remain associated with or lay in close proximity of transcription sites where they contribute to the assembly of large nuclear bodies (1-2 µm in diameter) that we christened “nuclear stress bodies” or nSBs. SatIII RNAs have a major role in the formation of nSBs since they recruit to these nuclear districts a specific subset of RNA binding proteins that normally act in pre-mRNA splicing.
Our working hypothesis is that SatIII RNAs, in addition to affect the nuclear structure may control the expression program of specific genes acting at the post-transcriptional level. Their ability to sequester pre-mRNA processing factors, including splicing factor SF2/ASF, in specific nuclear domains, in fact, is likely to affect the alternative splicing program of sets of genes during the recovery from stress.

 




Figure 2. Heat-shocked HeLa cells stained with an oligonucleotide complementary to Satellite III RNAs. Nuclear stress bodies are detectable as bright green spots in the nucleus close to the nucleoli.




Figure 3. Schematic representation of the assembly/disassembly of nuclear stress bodies after stress treatment.


The aims of our research programs are: 1) to identify transcripts whose alternative splicing profile is affected by SatIII RNAs; 2) to identify additional proteins accumulating in nSBs; 3) to study the expression of SatIII sequences in unstressed cells; 4) to explore the possible role of these ncRNA in the higher-order organization of SatIII arrays and finally 5) to identify physiological/pathological conditions in which these RNAs are expressed in the organism.

References

  1. Valgardsdottir R, Chiodi I, Giordano M, Cobianchi F, Riva S, Biamonti G. (2005) Structural and functional characterization of noncoding repetitive RNAs transcribed in stressed human cells. Mol Biol Cell. 16:2597-604.
  2. Chiodi I, Corioni M, Giordano M, Valgardsdottir R, Ghigna C, Cobianchi F, Xu RM, Riva S, Biamonti G. (2004) RNA recognition motif 2 directs the recruitment of SF2/ASF to nuclear stress bodies. Nucleic Acids Res. 32:4127-36.
  3. Biamonti G. (2004) Nuclear stress bodies: a heterochromatin affair?
  4. Nat Rev Mol Cell Biol. 5:493-8.
  5. Rizzi N, Denegri M, Chiodi I, Corioni M, Valgardsdottir R, Cobianchi F, Riva S, Biamonti G. (2004) Transcriptional activation of a constitutive heterochromatic domain of the human genome in response to heat shock. Mol Biol Cell. 15:543-51.
  6. Denegri M, Moralli D, Rocchi M, Biggiogera M, Raimondi E, Cobianchi F, De Carli L, Riva S, Biamonti G. (2002) Human chromosomes 9, 12, and 15 contain the nucleation sites of stress-induced nuclear bodies.  Mol Biol Cell. 13:2069-79.
  7. Denegri M, Chiodi I, Corioni M, Cobianchi F, Riva S, Biamonti G. (2001) Stress-induced nuclear bodies are sites of accumulation of pre-mRNA processing factors. Mol Biol Cell. 12:3502-14.
  8. Chiodi I, Biggiogera M, Denegri M, Corioni M, Weighardt F, Cobianchi F, Riva S, Biamonti G. (2000) Structure and dynamics of hnRNP-labelled nuclear bodies induced by stress treatments. J Cell Sci. 113:4043-53.
  9. Weighardt F, Cobianchi F, Cartegni L, Chiodi I, Villa A, Riva S, Biamonti G.(1999) A novel hnRNP protein (HAP/SAF-B) enters a subset of hnRNP complexes and relocates in nuclear granules in response to heat shock. J Cell Sci. 112:1465-76

 

 


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