A secretion-enhancing cis regulatory targeting element (SECReTE) involved in mRNA targeting to the ER and its role in protein secretion

mRNAs encoding soluble secreted and membrane proteins (mSMPs) are targeted to the endoplasmic reticulum (ER) to facilitate translation, translocation and secretion. Therefore, we searched for RNA motifs that enhance ER targeting and association and its RNA-binding proteins (RBPs). We identified a nucleotide repeat motif present in mSMPs and demonstrated its role related to mRNA stability, localization and the enhancement in protein secretion. Called SECReTE, for secretion-enhancing cis regulatory targeting element, this motif consists of triplet nucleotide repeats with a pyrimidine every third base. Mutational analyses using secreted proteins revealed that SECReTE addition enhances protein synthesis and secretion, while its removal lessens them. To further understand how SECReTE acts, we are identifying which RBPs are its partners in yeast. We examined RBPs that bind mSMPs - notably Bfr1, Whi3, Puf1, Puf2, Scp160, and Khd1. Considering that removal of the SECReTE might confer a detectable phenotype, we used HSP150, which encodes a secreted protein required for resistance to calcofluor white (CFW). The construction of a modified HSP150 mRNA, HSP150SECReTE(+), to increase the number of motif repeats without changing the amino acid sequence, made HSP150SECReTE (+) cells more resistant to CFW than WT. We deleted the RBPs to assess which could be SECReTE’s binding partner(s) and found that the deletion of WHI3 or KHD1 eliminated the differences between WT and HSP150(+) strains on CFW-containing plates. This suggests Whi3 and Khd1 bind HSP150 mRNA via SECReTE. We are now performing SECReTE-mediated RBP precipitation, followed by mass spectrophotometry to confirm which RBPs bind to SECReTE.