Caputi, Massimo

Serine/Arginine splicing factor 1 (SRSF1) is an RNA-binding protein (RBP) with multiple functions in RNA biogenesis. SRSF1 plays a prominent role in oncogenesis, immune function, and response to several physiological stimuli. To date, the role of SRSF1 as a regulator of mRNA splicing has been largely considered the main mechanism driving its biological functions and its role in disease. We have now characterized SRSF1’s role in Human Immunodeficiency Virus Type I (HIV-1) transcription. SRSF1 interacts with the 7SK small nuclear ribonucleoprotein (snRNP) to mobilize and activate the positive transcription-elongation factor (P-TEFb), which is then positioned on the HIV-1 promoter to increase the processivity of RNA polymerase II (RNAPolII) and promote the release of the negative regulators of transcription DSIF/NELF. Next, we defined the role of SRSF1 in the transcription of cellular genes utilizing an RNA sequencing (RNASeq) time course approach was used to detect changes in the transcriptome in response to SRSF1 overexpression. RNASeq data analysis revealed a subset of genes that were upregulated in response to SRSF1 overexpression. Nuclear run-on and qPCR assays experimentally validated 28 of these genes.

The largest barrier to treatment of HIV-1 infection is the establishment of a viral reservoir constituted mostly by quiescent latently infected CD4+ T cells. This reservoir is formed through two processes: i) the infection of resting CD4+ T cells; both naïve and memory, ii) the infection of activated CD4+ T cells which then become quiescent infected cells. One goal of this project was to understand the gene expression changes occurring in naïve CD4+ T cells following activation and subsequent HIV-1 infection and how this may contribute to the establishment of a latent infection in these cells. Utilizing RNA-Seq and a series of validation assays we have identified several genes which are regulated in opposite directions during activation versus infection which we termed DEOC genes. The DEOC genes include a group of physically- and functionally-associated proteins which are key regulators of T cell activation, the cell cycle, cellular proliferation, and cellular quiescence, suggesting that modulation of these DEOC genes may help transition the infected-activated cell from an activated state to a quiescent/resting state to induce a latent infection.

Serine/Arginine splicing factor 1 (SRSF1), a member of the Serine/Arginine rich (SR) RNA-binding proteins (RBPs) family, regulates mRNA biogenesis at multiple steps and is deregulated in cancer and autoimmune diseases. Preliminary studies show that members of the SR protein family play a role in cellular transcription. We investigated SRSF1’s role in cellular gene transcription utilizing time-course RNA-Seq and nuclear run-on assays, validating a subset of genes transcriptionally regulated following SRSF1 overexpression. Pathway analysis showed that genes in the TNF/IL17 pathways were enriched in this dataset. Furthermore, we showed that MyD88, a strong activator of TNF transcription through transcription factors NF-κB and AP-1, is a primary target of SRSF1’s transcriptional activity. We propose that SRSF1 activates the transcription factors NF-κB and AP-1 through MyD88 pathway. SRSF1 overexpression regulates several genes that are deregulated in malignancies and immune disease, suggesting a role for SRSF1’s transcriptional activity in oncogenesis and immune response regulation.

The Bel family of genes are fundamental to the apoptotic mechanism. Bcl-x a
member of this family, is alternatively spliced to create two main isoforms a long
(Bcl-xL) and a short (Bcl-xS) variant. The long form exhibits anti-apoptotic activity,
while the short form favors apoptosis. The proper balance of expression of these two
isoforms is crucial for several developmental processes such as thymic selection and
neural reshaping. A number of cancer types have been shown to over-express the long
form, thereby granting them some protection from apoptosis. To study the
transcriptional and post-transcriptional mechanisms regulating gene expression, the
Bcl-x gene has been utilized. A complex mini-gene construct has been create in order
to monitor the effects that promoter sequences, 5'UTR and 3'UTR's have on mRNA
splicing, RNA export, stability and translation. Abundant evidence exists indicating
that RNA processing events such as transcription, splicing and export are coupled, yet
the mechanisms and factors involved in regulating these processes are poorly
understood. The mini-gene is identical to the endogenous gene with the exception of a deletion to the 50Kb intron and the addition of a tag to differentiate the mini-gene
product from the endogenous mRNA and protein. This novel system allows for the
study of transcriptional and post-transcriptional mechanisms regulating gene
expression from RNA biogenesis on to the protein level.

The RRE is a sequence of the HIV genome which is required for the export of the
unspliced mRNA from the nucleus to the cytoplasm. Previous studies show that RRE
on the HIV mRNA binds directly to Rev which then interacts with Ran and CRM 1 to
form an export complex. Our results indicate that Ran can interact with the RRE in
the absence of Rev and CRM 1 but in the presence of other factor(s) present in the nuclear extract. Ran-GEF or RCC 1 seems to be a potential mediating factor. Our
results suggest that Ran binds directly to RCC 1 and that the binding is disrupted by
addition of excess nucleotides and magnesimn. Our suggestion is that Ran and RCC 1
are members of an alternate export pathway present in the HIV. Our observation that the binding is nonspecific makes us speculate that this export pathway may be present
in other cell types as well.