Michel Bellini

B.S., Pierre and Marie Curie University, Paris, France (Biochemistry)
M.S./Ph.D., Pierre and Marie Curie University, Paris, France (Molecular and Cellular Developmental Biology)
Postdoc., Carnegie Institution of Washington

Organization of the nucleus; chromosomes and other nuclear organelles; RNA transcription and processing; ribonucleoproteins, nucleo-cytoplasmic trafficking.

Background and significance

During the last two decades, the cell nucleus has emerged as a complex organelle highly organized into many distinct functional domains, and a true challenge for the 21rst century is to determine how these various domains cooperate to orchestrate the nuclear functions.  Among these functions, pre-mRNA splicing is critical for a correct proteome expression in all eukaryotic cells, and the importance of this RNA maturation event in the regulation of fundamental biological processes is highlighted by the ever-growing list of human disorders associated with splicing defects. Pre-mRNA splicing requires the five small nuclear ribonucleoprotein particles (U1, U2, U4, U5 and U6 snRNPs), each consisting of a small nuclear RNA (snRNA) and a cortege of associated proteins.  The snRNPs, along with over 300 other factors assemble onto pre-mRNAs to form the spliceosome, which is the dynamic enzyme that excises introns and ligates exons to produce mature mRNAs.  In the current view, spliceosomal assembly is tightly coupled to RNA transcription.  Accordingly, the snRNPs are known to associate with nascent RNA polymerase (RNAP) II transcripts.  Data on the spatial and temporal recruitment of splicing factors onto a template pre-mRNA abound in vitro.  However, little is still known in vivo about the essential characteristics of an snRNP that regulate its association with nascent transcripts and function in co-transcriptional splicing.  Since the interaction of snRNPs with pre-mRNAs is a prerequisite for the assembly of the spliceosome, a lack of understanding of how it is controlled in cells is a critical problem to overcome in order to fully understand the processes that modulate spliceosomal activity.

My long-term goal is to determine how the spliceosome assembly onto nascent transcripts is regulated in cells.  My objective for the coming years is to determine the inherent properties of snRNPs required for their interactions with nascent pre-mRNAs and their co-transcriptional splicing activity.  The central hypothesis is that recruitment of snRNPs to elongating transcripts is independent of the co-transcriptional assembly/activity of spliceosomes (Patel et al., 2007). I am particularly well prepared to undertake the proposed research because of our recent development of several in vivo assays that enable the study of co-transcriptional splicing in situ.  In addition, and to the best of my knowledge, my group is the only one to have obtained live images of individual active RNAPII transcription units where nascent transcripts are readily distinguishable (Patel et al., 2008).

In cultured somatic cells, efforts to monitor sites of transcription are hindered by the dense grouping of somatic nuclear structures and the low resolution of light microscopy, which is inadequate to resolve fine chromatin structures for a detailed analysis of RNA transcriptional events. To overcome these limitations, I employ the amphibian oocyte nucleus, which contains chromosomes and organelles that are an order of magnitude larger than their somatic counterparts and, thus, are readily observable with transmitted light.  In the nucleus of a Xenopus laevis oocyte, chromatin is organized within 18 lampbrush chromosomes (LBCs) and ~2000 non-chromosomal nucleoli.  LBCs are best described as highly extended diplotene bivalent chromosomes, and their characteristic resemblance to a test-tube brush comes from the fact that each homologue consists of a heterochromatin axis from which are projected numerous pairs of lateral loops.  These loops correspond to active transcriptional sites by RNAPII and consist of euchromatin fibers surrounded by nascent RNP fibrils.

To study the intranuclear trafficking of snRNPs in vivo, we exploited the fact that microinjected fluorescent snRNAs rapidly assemble into functional snRNPs (reviewed in Patel and Bellini, 2008).  While other groups have been using the same strategy previously, my laboratory is the first one to report their association with the LBC loops.  Interestingly, these data translated into a new and unique assay to begin investigate the mechanisms that regulate the association of snRNPs with active transcriptional units (Patel et al. 2007).

In addition, we established a new in vivo splicing assay to test the functionality of these associations.  Briefly, we demonstrated that the protein Y14, a core component of the Exon Junction Complex (EJC), associates with chromosomal loops in a splicing-dependent way (Patel et al. 2007).  Since EJCs are deposited by the spliceosome to mark exon-exon junctions, it demonstrates that pre-mRNA splicing occurs directly on the chromosomal loops.  We are currently using this functional assay to investigate which property of an snRNP is critical for its co-transcriptional splicing activity.

The association of Y14 with chromosomal loops also shows that EJCs engage nascent transcripts before their release from sites of transcription.  Following their deposition on transcripts, EJCs are involved in recruiting components of the mRNP export machinery. We recently found that the export factor Aly/REF is also present on chromosomal loops and its association with pre-mRNAs, like that of Y14, depends on splicing.  We are currently investigating other export factors such as the protein Tap.  Collectively, our data strongly support a model where the synthesis of a spliced, export-competent mRNP occurs directly on transcriptional units.

Concurrently, we have started to analyze the loop distribution of several other processing factors, such as hnRNPs A1, A2, Q, L and G, as well as nuclear factor 7 (Beenders et al., 2007).  While several of these proteins were initially described as “mRNA chaperones”, they were recently shown to have a direct role in the regulation of splicing.  We recently showed that the recruitment of hnRNP G, which antagonizes the role of the splicing factor Tra2ß, to nascent RNP fibrils does not require its RNA Recognition Motif, but rather a discrete domain in its carboxyl terminus. The same may be true for other hnRNPs and this result highlights the need to determine in vivo the recruitment mechanisms of these factors in order to better understand how RNA processing is regulated co-transcriptionally.

Finally, it is essential to consider the overall structure of LBCs in order to fully understand the dynamics of the chromosomal loops. We have concentrated our initial efforts on factors involved in chromatin condensation and we showed that some of the condensins such as XCAPD2, but not all of them, are components of LBCs (Beenders et al. 2003).  Since loops correspond to chromatin region where the sister chromatids are not associated, we are also currently analyzing the chromosomal association dynamics of several subunits of the cohesin complex, which is involved in sister chromatid cohesion (Austin et al., 2009).  Lastly, we demonstrated that the methyl-cytosine binding protein 2 (MeCP2) distributes within LBCs in a unique axial pattern that suggests a role in loop formation and/or maintenance.  The interest in MeCP2 lies within its ability to recruit histone deacetylases to methylated DNA and/or its possible involvement in chromatin loop formation in somatic systems to regulate gene expression.

In summary, using our newly established assays, we obtained results that reveal new levels of regulation in the pre-mRNA maturation pathway in vivo.  In particular we obtained original sets of data on the mechanisms that regulate the interaction of snRNPs with nascent transcripts and other organelles such as CBs and IGCs.  These results suggest a critical staging of the various RNA processing factors directly onto the nascent RNP particles for splicing.  In addition, we demonstrated a co-transcriptional recruitment of Y14 and Aly/REF, which underscores a complex interplay between transcription, processing, and export on the chromosomal loops.  Finally, we began to investigate the mechanisms that regulate at the chromatin level the formation and/or maintenance of the loops.

Future directions

To date the standard paradigm for analyzing transcriptional and RNA processing mechanisms has relied heavily on biochemical assays.  While these in vitro approaches are critical to establish the molecular mechanistics of pre-mRNA splicing, they usually fail to recapitulate the complexity of the intra-nuclear compartment.  In particular, the functional interactions of snRNPs with various nuclear organelles, including chromosomes, cannot be studied.  My research program is aimed at moving analysis to the lampbrush chromosome system where a direct visualization of individual active transcriptional units is possible.

National Academies Education Mentor in the Life Sciences (2012)
National Premedical Honor Society Professor of the Year (2005)
La Ligue contre le cancer Fellow (1991-1993)

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