Identification and characterization of RNA target sequences of plant RNA-binding proteins
Abstract
Eukaryotic cells express a variety of RNA-binding proteins that have important roles in post-transcriptional control of gene expression, including RNA maturation, transport, stability/decay and translation. Here, in vitro RNA target selection was used in an effort to identify the consensus RNA target sequences for members of two groups of plant RNA-binding proteins: the PUF family of RNA-binding proteins, and two peroxisomal matrix proteins that have previously been shown to bind to RNA.
Plant genomes encode large families of PUF proteins and these show variability in their predicted Puf repeat number, organization, and amino acid sequence. RNA target identification using in vitro selection identified high confidence data for four Arabidopsis PUF (APUM) proteins. Three of these proteins bound to classical PUF RNA target sequences. However one, the nucleolar targeted APUM23, bound to a novel PUF RNA binding sequence. The APUM23 binding sequence is ten nucleotides in length, containing a centrally located UUGA core element, and a preferred cytosine at nucleotide position 8. These RNA sequence characteristics differ from those of other PUF proteins, as all natural PUFs studied to date bind to RNAs that contain a conserved UGU sequence at their 5’ end and lack specificity for cytosine. The preferred 10-nucleotide sequence bound by APUM23 is present within the 18S rRNA sequence, supporting the known role of APUM23 in 18S rRNA maturation.
The RNA in vitro selection results for the peroxisomal matrix proteins malate dehydrogenase (pMDH) and multifunctional protein (pMFP) demonstrated that pMDH (but not pMFP) possesses sequence-specific RNA binding activity. pMDH bound with high affinity to two RNA consensus sequences, one that consisted of a triple-repeat of a four nucleotide sequence, and the other sequence was a nine-nucleotide palindrome.
This research provides insight into the novelty of plant RNA-binding proteins and their target RNA sequences, and sheds light on the role of these two classes of proteins in post-transcriptional control of gene expression. It reveals that APUM23 could provide an advanced structural backbone for Puf repeat engineering and target-specific regulation of cellular RNAs. This work also builds upon existing evidence for authentic RNA binding activities for some metabolic enzymes.
Description
Keywords
Botany, Biology--Molecular
Citation
Zhang, C. (2016). Identification and characterization of RNA target sequences of plant RNA-binding proteins (Doctoral thesis, University of Calgary, Calgary, Canada). Retrieved from https://prism.ucalgary.ca. doi:10.11575/PRISM/26418