tRNA-Derived Fragments (tRFs): Emerging New Roles for an Ancient RNA in the Regulation of Gene Expression

Round  1

Reviewer 1 Report

The emergence of mass sequencing has revealed a previously unexpected array of small RNAs derived from larger non-coding RNAs (such as tRNAs described here). Given their abundance and variety, it is an important question as to what function (if any) they perform. Quite a lot is now known regarding their abundance, production and possible function, though much remains uncertain. Hence, I believe this to be a timely and important topic for review.

The review is well written and I have only minor comments for possible alteration / consideration by the authors:

1) On line 74-76, can  the authors check that the correct cleavage of the appropriate arms are described in order to generate 5' and 3' tRFs as the description and the diagram in Figure 1 do not seem to match.

2) On line 102, the authors make a critical point (to my mind at least) stating that "correct modification of tRNA lowers conformation flexibility and increases thermal stability", This suggests what might (perhaps) be THE MAJOR explanation for their existence: a check of correct tRNA structure/function and subsequent cleavage of incorrect forms // the loss of tRNAs to halt translation under stress. That is not to say that these fragments are not interesting...merely that their presence (though individually of limited function) may point to itself an important process. When the authors make the case for their important functions (lines 170-179), this point is not addressed. Do the authors feel it appropriate to clearly state this possibility?

3) The abundance of such fragments causes an annotation headache as the authors describe (line 205). Could the authors slightly expand on their opinions how to address this? Are these 8 tRFs really "incorrectly annotated" also as miRNAs?

4) Could the authors also provide a small commentary regarding other related events occurring with other ncRNAs? snoRNAs, rRNAs, YRNAs, vtRNAs etc all show similar sorts of fragments? In terms of biogenesis or function, are tRFs actuallly something different or merely one example of a wider set of small RNA processing events?

Author Response

Author Response File: Author Response.pdf

Reviewer 3 Report

This review by Keam and Hutvagner touches a very timely and currently also quite hot topic of RNA biology, namely the potential cellular role(s) and the biogenesis of tRNA-derived fragments. The center piece of this review article in my opinion is a very nice overview picture (Fig 1) graphically highlighting the current knowledge of this emerging class of ncRNA regulators in a clear manner.

Unfortunately the text of the review does not reach the same level of clarity and sophistication (yet). For most of the parts it reads like a rather raw and preliminary draft version of a review article. Here are the major points that merit attention:

a)  for me it is unclear why the authors refrain from including tRNA halves in their review manuscript. Stress-induced tRNA halves are among the most extensively characterized tRNA-derived pieces and it would make much sense to include them in this manuscript as well. This inclusion would make this review much more comprehensive.

b) Chapter 2 (page 1): when describing tRNA maturation it is important to also discuss splicing of tRNA introns. Especially since some tRNA-derived fragments appear to be processed from pre-tRNA transcripts harboring introns (e.g. Hanada T. et al., Nature 2013).

c) page 1; line 42: it is incorrect that 3’ CCA ends are never encoded in genomes. tRNA genes in Gram negative bacteria do encode the 3’ CCA tRNA termini.

d) page 5, lane 129: “…associated with polysomes in HeLa…” is a quite sloppy wording. It should read “HeLa cells”.

e) the authors frequently use the phrases “lower organisms” and “higher organisms” throughout the text. From an evolutionary standpoint and strictly speaking the words “higher” and “lower” do not make much sense in this context. All extant organisms are as “low” or as “high” as all other extant organism regardless if they are pro- or eukarya. I suppose the authors meant more or less complex organisms.

f) page 7, line 229: which ribosomal subunit are the authors referring to here?

g) page 7, line 242: the citation to Haussecker et al 2010 is missing.

h) page 8, line 267: it should read “…….highly proliferative cells such as cancer cells.”

i) page 8: The paragraph dealing with tRFs as potential biomarkers should be removed from chapter 3 (Functionality of tRFs), since being a marker for a disease does not mean that the tRFs have a biologic function.

j) page 6; line 193: “has” needs to be replaced by “have”

k) References: almost all references carry mistakes with lower and upper case letters.  

Author Response

Author Response File: Author Response.pdf