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Biochemical and genetic analysis of a mouse DEAD‐box protein homologous to yeast Mtr4p
Author(s) -
Bestul Andrew Joseph,
Wang Xuying,
Anderson James T.,
Munroe Stephen H.
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.21.6.a1006
Mtr4p is an essential DExH‐box protein and putative RNA helicase that has been implicated in the export and processing of nuclear RNAs in yeast. Recently, Mtr4p has been identified as a component of a novel RNA surveillance complex in yeast known as the TRAMP complex. The TRAMP complex comprises three proteins: Trf4/5p, Air1/2p and the Mtr4p. Trf4p (also Pap2p) and Trf5p are poly(A) polymerases that are required for exosome‐mediated degradation of diverse non‐coding RNAs in the cell nucleus. 1 The Air 1/2 proteins are zinc finger proteins that may serve to bind RNA undergoing modification by the TRAMP complex. Mtr4p is essential for viability in yeast. It is also remarkably conserved in other eukaryotes. We have identified a mouse homolog that is 51% identical and 70% similar to Mtr4p in yeast over its entire length of 1040 amino acids. In order to better understand the function of this protein we have used siRNAs targeted against mouse Mtr4 mRNA to knockdown expression of mRNA in neuro 2a embryonic mouse cells. We have also cloned the mouse protein in a bacterial expression vector. Antibodies raised against recombinant mouse Mtr4p will be used to confirm the efficacy of these knockdowns on protein expression. Further experiments will be directed at comparing the biochemical properties of the mouse protein with those of yeast Mtr4p. siRNA knockdowns will be used to determine if its function as well as its structure is conserved between yeast and mammals.