# MUSVTS110:  THERMOSENSITIVE RNA SPLICING IN INTACT CELLS

> **NIH NIH R01** · UNIVERSITY OF TX MD ANDERSON CAN CTR · 1988 · $119,391

## Abstract

The objectives of this research proposal are to exploit the novel way in
which expression of the transformed phenotype is modulated in cells
infected with the MuSVts110 murine sarcoma virus as a means to study the
molecular basis of RNA splicing.  MuSVts110 infected cells (6m2 cells) are
transformed by the P85gag-mos, a virus-encoded gag-mos polypeptide
synthesized from a spliced 3.5KB viral mRNA.  The splice events which
produce this RNA occur in infected cells only at growth temperatures of 33
degrees C (or lower).  We plan to make advantage of the fact that although
MuSVts110 viral transcripts (4kb) are efficiently synthesized at 39 degrees
C, they are not spliced unless the growth temperature is lowered to at
least 33 degrees C.  Thus, we plan to dissect successive events in the
mechanism by which this viral RNA is spliced in intact cells under
physiologic conditions by shifting 6m2 cells from 39 degrees C to 33
degrees C for specified periods of time during the splicing process.  By
using S-1 nuclease protection analysis, primer extensive and nucleotide
sequencing analyses with specific DNA reagents capable of measuring splice
donor site cleavage, 'lariat' formation within the intron, events near the
splice acceptor site, and ligation of the 5' and 3' exons, we plan to
define splicing of this RNA in detail.  Additionally, we plan to
investigate the structural basis for and constraints on thermosensitive RNA
splicing by altering the structure of sequences in and around the 'intron'
in MuSVts110 DNA by, for example, site-directed mutagenesis, removal or
additional of restriction fragments or digestion with processive enzymes
such as BAL-31.  The splicing properties of these altered viral DNAs will
be studied in vitro after their introduction into, for example, one of the
SP6 family of plasmid vectors and those that prove interesting will be
transfected into NRK or 3T3 cells to study their splicing properties in
vivo.  Preliminary evidence indicates that in MuSVts110 revertant cells
capable only of splice donor site cleavage that this is a thermosensitive
process, suggesting that introducing alterations into the intron of viral
DNA obtained from this cell line and then measuring donor site cleavage
will provide a rapid assay for the effects of structural alterations on RNA
splicing.  If, as is our current expectation, we find that is the structure
of the MuSVts110 intron is responsible for thermosensitive RNA splicing, we
plan to construct an expression vector capable of the temperature-sensitive
expression of cloned genes in euraryotic cells.

## Key facts

- **NIH application ID:** 3172520
- **Project number:** 5R01CA034734-06
- **Recipient organization:** UNIVERSITY OF TX MD ANDERSON CAN CTR
- **SAM.gov UEI:** S3GMKS8ELA16
- **Principal Investigator:** EDWIN C MURPHY
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 1988
- **Award amount:** $119,391
- **Award type:** 5
- **Project period:** 1983-06-01 → 1989-11-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/3172520

## Citation

> US National Institutes of Health, RePORTER application 3172520, MUSVTS110:  THERMOSENSITIVE RNA SPLICING IN INTACT CELLS (5R01CA034734-06). Retrieved via MORVS 2026-10-25 from https://api.morvs.ai/grant/nih/3172520. Licensed CC0.

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