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\DOI{10.5802/crbiol.138}
\datereceived{2023-10-13}
\dateaccepted{2023-10-23}
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\dateposted{2024-01-18}
\datepublished{2024-03-29}
\begin{document}

\begin{noXML}

%\makeatletter
%\def\TITREspecial{\relax}
%\def\cdr@specialtitle@english{A tribute to Fran\c{c}ois Gros, a founding father of molecular biology}
%%\def\cdr@specialtitle@french{Un hommage \`a Fran\c{c}ois Gros, un des p\`eres de la biologie mol\'eculaire}
%\makeatother

\title{Memories of a young student: the early days of splicing
regulation with Fran\c{c}ois Gros}

\alttitle{Souvenirs d'un jeune \'{e}tudiant : les d\'{e}buts de la
r\'{e}gulation de l'\'{e}pissage avec Fran\c{c}ois Gros}

\author{\firstname{Domenico} \lastname{Libri}\CDRorcid{0000-0001-6728-0594}}
\address{Institut de G\'{e}n\'{e}tique Mol\'{e}culaire de Montpellier,
Montpellier, France}
\email{domenico.libri@igmm.cnrs.fr}

\keywords{\kwd{Memories}\kwd{Alternative splicing}\kwd{Muscle
differentiation}\kwd{History of muscle protein genes}}

\altkeywords{\kwd{Souvenirs}\kwd{\'{E}pissage
alternatif}\kwd{Diff\'{e}rentiation musculaire}\kwd{Histoire des
g\`{e}nes de prot\'{e}ines musculaires}}

\def\thanksname{Note}
\thanks{This article follows a symposium held on 25 April 2023 at the
Institut Pasteur in tribute to Fran\c{c}ois Gros.}

\begin{abstract} 
I joined the laboratory of Fran\c{c}ois Gros as a young student in the
mid-1980s and worked on the characterization of the $\upbeta$-tropomyosin
gene in chicken and the regulation of alternative splicing of its
transcript, under the supervision of Marc Fiszman. In particular, I was
interested in how secondary structures of the RNA influence the
recognition of exons specifically used in muscle cells. I will recall a
few memories on how interacting with Fran\c{c}ois on this project
shaped my perception of the scientific process and of the relationships
between models and data. Later I worked on many aspects of RNA biology,
from transcription to mRNP biogenesis and non-coding RNAs.
%\vspace*{-3pt}
\end{abstract}

\begin{altabstract}
J'ai rejoint le laboratoire de Fran\c{c}ois Gros en tant que jeune
\'{e}tudiant au milieu des ann\'{e}es 1980 o\`{u} j'ai travaill\'{e}
sur la caract\'{e}risation du g\`{e}ne de la $\upbeta$-tropomyosine chez le
poulet et sur la r\'{e}gulation de l'\'{e}pissage alternatif de son
transcrit, sous la supervision de Marc Fiszman. En particulier, je me
suis int\'{e}ress\'{e} \`{a} la mani\`{e}re dont les structures
secondaires de l'ARN influencent la reconnaissance des exons
sp\'{e}cifiquement utilis\'{e}s dans les cellules musculaires.
J'\'{e}voquerai quelques souvenirs concernant la fa\c{c}on dont
l'interaction avec Fran\c{c}ois sur ce projet a fa\c{c}onn\'{e} ma
perception du processus scientifique et des relations entre les
mod\`{e}les et les donn\'{e}es. Par la suite, j'ai travaill\'{e} sur de
nombreux aspects de la biologie de l'ARN, de la transcription \`{a} la
biogen\`{e}se des mRNP et aux ARN non codants.
\end{altabstract}
\begin{DefTralics}
\newcommand{\upbeta}{\beta}
\end{DefTralics}
\maketitle

\vspace*{8pt}

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\xsection{}

I joined Fran\c{c}ois' laboratory in November 1984, coming from the
University of Pisa, as part of the exchange program between the Scuola
Normale of Pisa and the Ecole Normale Sup\'{e}rieure (ENS). This was
after completing my third year of University, which today would be
equivalent to a Master 1 degree. At the time, Fran\c{c}ois was leading
the Unit of Biochemistry at the Institut Pasteur and was also directing
a laboratory at the Coll\`{e}ge de France. The Unit of Biochemistry
housed several research groups, primarily focused on the biochemistry
of muscle, and later, muscle development: the groups of Marc Fiszman,
Robert (Bob) Whalen and Margaret Buckingham, who a few years later
became independent and founded her own unit in the same department.
Initially, the three groups were interested in the biochemistry and
expression of contractile proteins, with Bob and Margaret focusing more
on myosins and their mRNAs, \mbox{respectively}, and Marc on
tropomyosin. I had been accepted to work in Marc's group. 

\break
Marc had set up a nice cellular system whereby differentiation of
chicken myoblasts infected by a thermosensitive mutant of the Rous
sarcoma virus could be induced by shifting the culture temperature to
42~\textdegree C~\cite{1}. In Marc's group the expression of
tropomyosin isoforms induced specifically during differentiation was
studied biochemically. 

\begin{figure*}
{\vspace*{4pt}}
\includegraphics{fig01}
{\vspace*{7pt}}
\caption{\label{fig1}RNA secondary structure proposed to sequester exon
6B of the chicken tropomyosin gene to prevent its inclusion in
undifferentiated cells (Mb). A scheme of the exonic organization of the
gene in this region is shown in the bottom.} 
{\vspace*{4pt}}
\end{figure*}

But the molecular biology era had started with the isolation of cDNAs
that could be used for characterizing the many different mRNAs coding
for contractile proteins, for analyzing gene expression
and---importantly---for the characterization of genes. Articles had
been published by the group of Margaret on the structure of the myosin
light chain genes~\cite{2,3}.

In Marc's lab my task was to isolate the gene coding for
$\upbeta$-tropomyosin in chicken: we had a cDNA clone provided by David
Helfman and in Marc's group the expression of the isoforms of chicken
tropomyosin had been extensively studied during muscle development at
the protein level. When we determined the structure of the gene we
discovered that it was alternatively spliced, with two couples of exons
used in a mutually exclusive manner, exon 6A and 9B in myoblast, 6B and
9A in myotubes. The big question was then how the ``right'' exons are
chosen.

At that time, the understanding of splicing regulation was in its early
stages. The discovery that genes were composed of exons and introns had
occurred less than a decade earlier~\cite{4,5} and many examples of
alternatively spliced genes were emerging, starting in the early and
mid 80s~\cite{6}.

While there was much speculation and research on how the choice of
alternatively spliced exons was made, the underlying mechanisms
remained largely unknown. This was the challenge we faced. One of my
best souvenirs in Fran\c{c}ois' laboratory revolves around these
studies, and reflects the great oversight he provided, even from a
somewhat distant eye!

I had analyzed the sequence of the alternatively spliced region around
exons 6A and 6B using the available tools of the time, such as early
versions of the RNA folding algorithm created by Zuker~\cite{7}, now
called Mfold~\cite{8}. I had proposed a model suggesting that exon 6B
could not be used in myoblasts because it was sequestered within a
secondary structure of the primary transcript~\cite{9}. Upon
differentiation, an alternative secondary structure would form around
the other exon (6A), simultaneously freeing the muscle-specific exon
and sequestering the exon used in undifferentiated cells
(Figure~\ref{fig1}).

I was very proud of my model, the demonstration of which, in my
naiveness, I had taken for granted. Around that time the group of
Bernard Dujon had joined the Institute. I remember seeking the opinion
of Alain Jacquier, an RNA expert in Bernard's team and later on an
inspirational friend. Alain was very skeptical about the possibility
that such a structure would form in the cell, which of course left me
in deep despair. I remember walking back to the lab, clutching my now
seemingly useless model, and meeting Fran\c{c}ois, who read the
disappointment on my face. Fran\c{c}ois was a very busy person, I was
not even sure he knew who I was. But, of course, this encounter was a
bit like the branch you grab when falling off a cliff, and I started to
explain confusedly the reason of my despair. I certainly did not know
that Fran\c{c}ois had fallen into the ``magic RNA potion'' in his
youth: ``When Monod asked me what I would like to work on, now that I
was in his lab\,\ldots\ my decision was taken. I would, from then on
become an RNA biochemist, an RNA man! Nothing concerning RNA and its
role in protein \mbox{synthesis} should be \mbox{foreign} to me.''~\cite{10}.
Fran\c{c}ois invited me into his office and patiently listened to my
story. When I was done, he thought for a couple of minutes, and, of
course, agreed with Alain (\ldots\,falling down the cliff again). He
said: ``Domenico, ne tombez pas amoureux d'un mod\`{e}le, t\^{o}t ou
tard il vous d\'{e}cevra (don't fall in love with a model, sooner or
later it will disappoint you)''---I like to think he was wittily
playing with the double meaning of the word ``model''. But then he
added: ``\ldots\,mais soyez pers\'{e}v\'{e}rant pour le d\'{e}montrer
(but be perseverant in proving it''.

This was of course the net that saved my fall. Many mutations and
compensatory mutations later, the model was partially proven~\cite{11},
and secondary structures of the primary transcript are now believed to
have important roles in alternative splicing~\cite{12,13,14,15}.

I like to think of this anecdote as nicely summarizing Fran\c{c}ois'
legacy. We should be dreamers, sometimes visionaries, persistent in
pursuing demonstrative science, but never in love with models. I am
certain that I interpret the thoughts of many colleagues and friends of
that period in thanking Fran\c{c}ois for building the great and
supportive environment that made possible a very nice moment of science
in the Unit of Biochemistry of the Institut Pasteur.

\section*{Declaration of interests}

The authors do not work for, advise, own shares in, or receive funds
from any organization that could benefit from this article, and have
declared no affiliations other than their research organizations.

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