Although currently controversial, one potential contribution is elements in coding regions, sometimes referred to as downstream sequence elements (reviewed in Gonzalez et al. 2001), that might recruit Upf3 and Upf2 to mRNAs, and, if Upf2 and Upf3 are not removed by elongating ribosomes, might lead to triggering NMD after upstream termination.
The P. vivax, P. cynomolgi and P. knowlesi msp-9 genes encode proteins that range in calculated molecular mass from 80 to 107 kDa, have typical eukaryotic signal peptides and diverse repeated motifs present immediately upstream of their termination codon.
This region of the mRNA, termed the termination upstream ribosomal binding site (TURBS), is needed for the retention of post-termination 40S subunits [11].
In both organisms there is a preference for A in the position two bases upstream of the termination codon and U immediately downstream [ 14].
Such shifts have been reported by Tatyana Pestova and should be addressed and cited (Alkalaeva et al., Cell 2006) as they may explain why occupancy isn't always seen exactly 15 nt upstream at the termination codon.
Insertion of the reporter immediately upstream of the termination codon common to all transcripts yielded nuclear-localized GFP in multiple discrete components including nerve cells, muscle cells, the vulva, the hypodermal seam cells and the intestine, and from embryogenesis to the adult (WBID Expr9786/9837).
Two lines obtained from T-DNA mutant collections were characterised by sequencing from both T-DNA borders: SALK_102345, an insertion in the last exon of REV (At5g60690) upstream of the termination codon, and GABI-KAT line GK_302G09, an insertion affecting the second exon of CUC3 (At1g76420).
As can be seen in Figure 2b, the latter mutation greatly reduced expression of the VP2fluc product, supporting the idea that an interaction between the 18S rRNA and the mRNA just upstream of the termination-reinitiation site is required.
Motif 2, which lies some 15 20 nt upstream of the termination-reinitiation window (AUGA in FCV), is a second essential stretch of 4 8 nt but shows little sequence conservation amongst caliciviruses.
Notably, the increased expression of ORF transcripts in the rrp6Δ mutant is not due to defects in transcription termination from upstream loci, as the upstream expression level (defined as −100 to TSS) from these ORFs correlates poorly with the downstream expression levels (defined as TSS to +100).
Site-directed mutagenesis was used to insert two translation termination codons upstream of the C-terminal Met to prevent expression of this Met or the 6His tag so that CNBr cleaved product would result in release of a native sequence peptide from the KSI partner.
