scholarly journals To New Beginnings: Riboproteogenomics Discovery of N-Terminal Proteoforms in Arabidopsis Thaliana

2022 ◽  
Vol 12 ◽  
Author(s):  
Patrick Willems ◽  
Elvis Ndah ◽  
Veronique Jonckheere ◽  
Frank Van Breusegem ◽  
Petra Van Damme

Alternative translation initiation is a widespread event in biology that can shape multiple protein forms or proteoforms from a single gene. However, the respective contribution of alternative translation to protein complexity remains largely enigmatic. By complementary ribosome profiling and N-terminal proteomics (i.e., riboproteogenomics), we provide clear-cut evidence for ~90 N-terminal proteoform pairs shaped by (alternative) translation initiation in Arabidopsis thaliana. Next to several cases additionally confirmed by directed mutagenesis, identified alternative protein N-termini follow the enzymatic rules of co-translational N-terminal protein acetylation and initiator methionine removal. In contrast to other eukaryotic models, N-terminal acetylation in plants cannot generally be considered as a proxy of translation initiation because of its posttranslational occurrence on mature proteolytic neo-termini (N-termini) localized in the chloroplast stroma. Quantification of N-terminal acetylation revealed differing co- vs. posttranslational N-terminal acetylation patterns. Intriguingly, our data additionally hints to alternative translation initiation serving as a common mechanism to supply protein copies in multiple cellular compartments, as alternative translation sites are often in close proximity to cleavage sites of N-terminal transit sequences of nuclear-encoded chloroplastic and mitochondrial proteins. Overall, riboproteogenomics screening enables the identification of (differential localized) N-terminal proteoforms raised upon alternative translation.

2019 ◽  
Author(s):  
Sezen Meydan ◽  
James Marks ◽  
Dorota Klepacki ◽  
Virag Sharma ◽  
Pavel V. Baranov ◽  
...  

SUMMARYThe use of alternative translation initiation sites enables production of more than one protein from a single gene, thereby expanding cellular proteome. Although several such examples have been serendipitously found in bacteria, genome-wide mapping of alternative translation start sites has been unattainable. We found that the antibiotic retapamulin specifically arrests initiating ribosomes at start codons of the genes. Retapamulin-enhanced Ribo-seq analysis (Ribo-RET) not only allowed mapping of conventional initiation sites at the beginning of the genes but, strikingly, it also revealed putative internal start sites in a number of Escherichia coli genes. Experiments demonstrated that the internal start codons can be recognized by the ribosomes and direct translation initiation in vitro and in vivo. Proteins, whose synthesis is initiated at an internal in-frame and out-of-frame start sites, can be functionally important and contribute to the ‘alternative’ bacterial proteome. The internal start sites my also play regulatory roles in gene expression.


Biochemistry ◽  
2000 ◽  
Vol 39 (33) ◽  
pp. 10189-10195 ◽  
Author(s):  
Mamatha Damodarasamy ◽  
Mei Zhang ◽  
Krista Dienger ◽  
Francis X. McCormack

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 75-75 ◽  
Author(s):  
Jonathan J. Keats ◽  
Christopher A. Maxwell ◽  
Tony Reiman ◽  
Brian J. Taylor ◽  
Michael J. Mant ◽  
...  

Abstract Translocations involving the IgH locus are common genetic events in multiple myeloma (MM). A number of recurrent IgH translocations exist with t(11;14)(q13;q32) and t(4;14)(p16;q32) being the most common. These translocations predict for differential clinical outcomes, good versus poor, respectively. We have shown that ~70% of t(4;14) POS patients express the initially proposed target gene FGFR3. However, the t(4;14)POS/FGFR3NEG group of patients still fare poorly (P=0.01). Therefore, either the transformation event associated with t(4;14) is multifactorial or independent of FGFR3. The loss of FGFR3 expression is associated with the loss of der(14). However, der(4) is detectable in all t(4;14)POS patients at diagnosis and relapse, suggesting that it is biologically and clinically relevant. The der(4) chromosome is thought to result in the overexpression of MMSET. The genomic breakpoints associated with t(4;14) occur in the 5′ end of the MMSET locus. In 70% of patients (MB4-1), the breakpoints maintain the full length open reading frame of MMSET. In the remaining 30% (MB4-2 & MB4-3), the breakpoints occur downstream of the proper translation initiation site. Two principle transcripts originate from the MMSET gene. The first transcript initiates in the beginning of the MMSET locus and, as a result of alternative splicing of exon 12, produces either MMSET I or MMSET II. The second transcript initiates upstream of exon 10 and uses an alternative translation initiation site to produces RE-IIBP. MMSET I and II transcripts, produced by each breakpoint variant, and the RE-IIBP transcript, produced in all patients irrespective of breakpoint type, were cloned. Transcripts were C-terminally tagged with GFP and transiently transfected into HeLa cells. Anti-GFP immunoblots showed that all transcripts produced a protein product, even the MB4-2 and MB4-3 variants that utilize alternative translation initiation sites in exon 4 and 6, respectively. The wildtype/MB4-1 MMSET I and II constructs localized to the nucleus and were excluded from nucleoli. MMSET II is almost exclusively associated with chromatin while MMSET I localized diffusely. RE-IIBP localized primarily in cytoplasmic foci and to nucleoli. Unlike the full length MMSET proteins, the MB4-2/MB4-3 constructs localized to the nucleus but also localized in nucleoli. To determine if the N-terminus regulates the nuclear localization pattern, we cloned the N-terminal portion of MMSET, which is lost in the MB4-2 transcripts. As this construct localized to the nucleus and was excluded from nucleoli, therefore a domain required for the proper localization of MMSET is lost in the MB4-2/MB4-3 variants. Kinetic analysis of MMSET variants localized to the nucleoplasm shows that the association of MMSET II with chromatin is very stable, t1/2 130 sec, while the type II MB4-2 and MB4-3 breakpoint variants have reduced kinetics, t1/2 19 and 12 sec, respectively, suggesting a decreased stability of association. The reduction in kinetics is also seen in the type I variants. We verified the overexpression of RE-IIBP by quantitative RT-PCR on a panel of purified plasma cells and unpurified BMMC. RE-IIBP was overexpressed in t(4;14)POS patients, P=0.0009 and P=0.00006, respectively, making it the only overexpressed protein without altered function in all t(4;14)POS patients irrespective of FGFR3 expression or breakpoint type. Therefore, RE-IIBP may be of central importance to the poor outcome of t(4;14)POS MM patients.


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